Biomedical subjects

Ferenc Hudecz

Publications and source records attributed to Ferenc Hudecz.

At least 19 recordsLinked to original sources

PubMed2007

Disulfide bond rearrangement during regioselective oxidation in PhS(O)Ph/CH3SiCl3 mixture for the synthesis of alpha-conotoxin GI.

Rearrangement of disulfide bonds during the synthesis of alpha-conotoxin GI using PhS(O)Ph/CH(3)SiCl(3) oxidation procedure was observed. We have demonstrated that the protecting scheme (order of acetamidomethyl (Acm) and (t)Bu protecting groups) of the Cys residues as well as the reaction time influenced the ratio of the native and the mispaired compounds, while the temperature of the reaction mixture had no significant effect. However, in all cases the nonnative derivative was produced in high amount. The structure of the isomers was identified by the combination of enzymatic digestion and mass spectrometry measurements. We conclude that the air oxidation followed by the application of Tl(tfa)(3) for the regioselective formation of disulfide bonds leads up to the appropriate compound in the case of the synthesis of alpha-conotoxin GI, while the oxidation procedure using PhS(O)Ph/CH(3)SiCl(3) system resulted in the nonnative disulfide isomer.

Amino Acid Sequence↗

PubMed2006

Synthesis and spectroscopic properties of 4-ethoxymethylene-2-(1)-naphthyl-5(4H)-oxazolone-labeled fluorescent peptides.

Strategies for the preparation of new fluorescent oligopeptide conjugates labeled with 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone (naOx-OEt) at the N-terminal on solid support or in solution have been devised. These procedures are simple and easy to carry out by reacting naOx-OEt or N(alpha)-naOx-amino acid with side chain protected peptide chains attached to resins. The integrity of the N-alkyl bond was maintained even after the trifluoracetic acid or HF based cleavages procedures. Our data show that the naOx fluorophore is compatible with both Fmoc/tBu and Boc/Bzl methods and also suggest that naOx-amino acid could be utilized as building blocks for solid phase peptide synthesis. Comparative analysis of fluorescence properties of naOx-conjugates indicated that the spectral properties of the fluorophore do not change after incorporating into peptides. The compact size, the definite chemical reaction for its introduction in combination with the appropriate spectral features (e.g., intense emission, pH independent fluorescent characteristics, and beneficial photobleaching dose constant and rates) and with chemical and spectral stability, naOx-based labeling could be attractive for novel cellular fluorescent techniques (e.g., in laser scanning confocal FRET) to study peptide-protein and protein-protein interactions even in biological matrices.

Alkylation↗

PubMed2006

The effect of the structure of branched polypeptide carrier on intracellular delivery of daunomycin.

The conjugate of acid labile cis-aconityl-daunomycin (cAD) with branched chain polypeptide, poly[Lys(Glui-DL-Alam)] (EAK) was very effective against L1210 leukemia in mice. However, Dau attached to a polycationic polypeptide, poly[Lys(Seri-DL-Alam)] (SAK) exhibited no in vivo antitumor effect. In order to understand this difference we have performed comparative in vitro studies to dissect properties related to interaction with the whole body (e.g., biodistribution) from those present at cellular or even molecular level. We report here (a) the kinetics of acid-induced Dau liberation, (b) interaction with DPPC phospholipid bilayer, (c) in vitro cytotoxic effect on different tumor cells, and (d) intracellular distribution in HL-60 cells of polycationic (cAD-SAK) and amphoteic (cAD-EAK) conjugates. Fluorescence properties of the two conjugates are also reported. Our findings demonstrate that the kinetics of the drug release, intracellular distribution and in vitro cytotoxic effect are rather similar, while the effect on DPPC phospholipid bilayer and fluorescence properties of the two conjugates are not the same. We also found that the in vitro cytotoxicity is cell line dependent. These observations suggest that the structure of the polypeptide carrier could have marked influence on drug uptake related events.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2006

Influence of sequential oligopeptide carriers on the bioactive structure of conjugated epitopes: comparative study of the conformation of a Herpes simplex virus glycoprotein gD-1 epitope in the free and conjugated form, and protein "built-in" crystal structure.

Synthetic carriers play an important role in immunogen presentation, due to their ability of inducing improved and specific responses to conjugated epitopes. Their influence on the bioactive conformation of the epitope, though admittedly crucial for relevant in vitro and in vivo applications, is difficult to evaluate, given the usual lack of information on the complex conformational features determined by the nature of the carrier and the mode of ligation. Using the Herpes simplex virus glycoprotein D-1 epitope (Leu(9)-Lys-Nle-Ala-Asp-Pro-Asn-Arg-Phe-Arg-Gly-Lys-Asp-Leu(22)) as a model, we have performed a detailed conformational analysis on the free epitope peptide in solution and on three constructs in which the epitope was conjugated to sequential oligopeptide carriers {Ac-[Lys-Aib-Gly](4)-OH (SOC(4))} (through either a thioether or an amide bond; Ac: acetyl) and polytuftsin oligomers {H-[Thr-Lys-Pro-Lys-Gly](4)-NH(2) (T20)}, (through a thioether bond). The analysis of the epitope conformation in the parent protein, in carrier-conjugated and free form, suggests that the beta-turn structure of the -Asp(13)-Pro-Asn-Arg(16)- segment is highly conserved and independent of the epitope form. However, small conformational variations were observed at the C-terminal part of the epitope, depending on the nature of the carrier.

Amino Acid Sequence↗

PubMed2006

Synthesis, structure, and in vitro antitumor activity of some glycoside derivatives of ferrocenyl-chalcones and ferrocenyl-pyrazolines.

Some new glycosides of 3-ferrocenyl-1-(4'-hydroxyphenyl)-prop-2-en-1-one were prepared and transformed into the corresponding pyrazoline and pyrazole derivatives. Using methyl-hydrazine, formation of regioisomers was observed. DDQ was found to be a mild and efficient reagent for the pyrazoline-pyrazole dehydroaromatization process. The structure of the new compounds was proved by IR and NMR spectroscopy. The in vitro antitumor activity of the substances was investigated against human leukemia (HL-60) cells by the MTT method. Among these new compounds some chalcone derivatives (3 a, 3 b, 5 a, and 5 b) showed attractive in vitro antitumor effects on human leukemia cells. These molecules contained ferrocenyl moieties and a p-hydroxy-phenolic ring or a size-independent apolar substitution of that.

Antineoplastic Agents↗

PubMed2006

Synthesis of oligotuftsin-based branched oligopeptide conjugates for chemotactic drug targeting.

The synthesis and chemotactic properties of a new class of branched oligopeptide-based conjugates are described. Tetratuftsin derivatives containing chemotactic formyl tripeptides (For-MLF, For-NleLF or For-MMM) in branches were prepared by stepwise solid-phase peptide synthesis. The influence of the composition and ionic charge of the carrier-branched oligopeptide on the chemotactic behaviour of the conjugate was studied in Tetrahymena pyriformis. Conjugates with methotrexate (Mtx) as a drug component was also prepared. For this, a GFLGC spacer, cleavable by cathepsin B, was used. The spacer with N-terminal methotrexate was coupled to the chloroacetylated chemotactic carrier molecule by thioether bond formation. The chemotactic activity and cytotoxity of Mtx conjugates were also studied.

Amino Acid Sequence↗

PubMed2006

Synthesis of enzymatically resistant nociceptin-related peptides containing a carbamic acid residue.

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.

Animals↗

PubMed2005

Synthesis and biological studies of nociceptin derivatives containing the DTPA chelating group for further labeling with therapeutic radionuclides.

Nociceptin is an endogenous anti-opiate heptadecapeptide primarily interacting with the nociceptin (NOP) receptor. This neuropeptide-receptor system is involved in pain regulation, tolerance to and dependence on opiates as well as many other physiological and pathophysiological events. The role and mechanisms of nociceptin in pathological conditions is not clearly known yet. In an attempt to have a radiopharmaceutical labeled either with 99mTc or (111)In, we incorporated diethylenetriaminepentaacetic acid (DTPA) as chelator into the structure of [Arg14,Lys15]nociceptin(1-17)-NH2 at the epsilon-amino group of Lys15. Such a radiopeptide may be useful in imaging for diagnostical purposes. Preparation of the peptide ligands was carried out by solid phase synthesis. Two peptides containing DTPA were obtained and purified. The products were [Arg14,Lys(DTPA)15]nociceptin(1-17)-NH2 and its cross-linked dimer on the basis of mass spectrometric analysis. In (115)In3+ binding experiments the conjugates exhibited preserved indium ion chelating properties, indicating the potential use of radiolabeled DTPA-nociceptin derivatives as radiopharmaceutical. Biological properties of these compounds were studied in rat brain membrane preparations by radioligand binding, functional biochemical [35S]GTPgammaS binding assays and mouse vas deferens (MVD) bioassay. Besides the similar in vitro binding characteristics to nociceptin receptor, both of the DTPA-chelated compounds were more potent and efficient than nociceptin in functional biochemical and mouse vas deferens bioassays. Our further aim is to radiolabel these compounds in order to get a radiopharmaceutical which can be used diagnostically.

Animals↗

PubMed2005

Detection of citrate synthase-reacting autoantibodies after heart transplantation: an epitope mapping study.

Autoimmune mechanisms play an important role in the pathogenesis of allograft vasculopathy following heart transplantation, but the autoantigens involved have been only sparsely studied. Citrate synthase (CS) enzyme is a conserved molecule, and, as an important mitochondrial autoantigen, it is protected by the "immunological homunculus". Tissue destruction and alteration of the immune regulatory mechanisms can induce pathological immune response against CS in other autoimmune diseases. In our present study we aimed to detect CS-specific autoantibodies in heart transplant patients, therefore, prospective, randomised clinical tests were conducted on 33 heart transplant patients and compared with 130 healthy blood donors. The level and isotype of CS antibodies were detected by simple binding indirect enzyme-linked immunosorbent assay (ELISA). The epitope specificities of the autoantibodies were measured on synthetic overlapping peptide sequences of CS enzyme by an indirect multi-pin ELISA method. Mainly IgM isotype CS autoantibodies were found in healthy controls, while IgG was found at higher levels and frequency (four-times higher) in heart transplant patients. Autoantibodies of IgG isotype recognise different epitopes than do autoantibodies of IgM isotype, even within the same group and individual. New epitope-specific IgG and IgM isotype autoantibodies appeared in heart transplant patients when compared with the controls. Our findings suggest a possible role of CS-specific autoantibodies in the pathomechanism of allograft vasculopathy.

Antibody Specificity↗

PubMed2005

Partial D-amino acid substitution: Improved enzymatic stability and preserved Ab recognition of a MUC2 epitope peptide.

The stability of an immunogen against enzymatic degradation is considered an important factor for the design of synthetic vaccines. For our studies, we have selected an epitope from the tandem-repeat unit of the high-molecular-weight MUC2 mucin glycoprotein, which can be underglycosylated in case of colon cancer. In this study, we prepared a MUC2 peptide containing the PTGTQ epitope of a MUC2 protein backbone-specific mAb 996 and its derivatives. In these peptides, the N- and C-terminal flanking regions were systematically substituted by up to three d-amino acids. Peptides prepared by solid-phase synthesis were tested for their mAb 996 binding in competitive ELISA experiments, and their stability was studied in serum and lysosomal preparation. Our data show that the epitope function of peptide (15)TPTPTGTQTPT(25) is retained even in the presence of two d-amino acid residues at its N-terminal flanking region and up to three at its C-terminal flanking region (tpTPTGTQtpt). Also, this partly d peptide shows high resistance against proteolytic degradation in diluted human serum and in lysosomal preparation. These findings suggest that, by appropriate combination of structural modifications (namely, d-amino acid substitution) in the flanks of an Ab epitope, it is feasible to construct a synthetic antigen with preserved recognition properties and high stability against enzymatic degradation. Peptides tPTPTGTQTpt and tpTPTGTQTpt derived from this study can be used for immunization experiments and as potential components of synthetic vaccines for tumor therapy.

Amino Acid Sequence↗

PubMed2005

MALDI-TOF mass spectrometry of a combinatorial peptide library: effect of matrix composition on signal suppression.

The effect of matrix composition on signal suppression caused by a dominant compound under MALDI ionization was studied using the combinatorial TQTXT pentapeptide library as a model system. The peptide library is composed of 19 components with all proteinogenic amino acids except cysteine in position X. From these compounds, only the Arg peptide (TQTRT) was detected with sufficient intensity in the MALDI-TOF mass spectrum under typical MALDI conditions (CCA matrix). The analysis of a set of compounds utilized as different matrix components, additives and a cationizing agent revealed that the composition of the matrix is a critical point in signal suppression. Highly improved ion yields were achieved by using a CCA/DHB mixture as a matrix. The addition of K(+) as a cationizing agent to the CCA matrix resulted in MALDI-TOF mass spectra with relative ion intensities very similar to those obtained by electrospray ionization.

Combinatorial Chemistry Techniques↗

PubMed2005

Medium-sized peptides as built in carriers for biologically active compounds.

A growing number of oligopeptides of natural and/or synthetic origin have been described and considered as targeting structures for delivery bioactive compounds into various cell types. This review will outline the discovery of peptide sequences and the corresponding mid-sized oligopeptides with membrane translocating properties and also summarize de novo designed structures possessing similar features. Conjugates and chimera constructs derived from these sequences with covalently attached bioactive peptide, epitope, oligonucleotide, PNA, drug, reporter molecule will be reviewed. A brief note will refer to the present understanding on the uptake mechanism at the end of each section.

Amino Acid Sequence↗

PubMed2005

The effect of cyclization on the enzymatic degradation of herpes simplex virus glycoprotein D derived epitope peptide.

One linear and three cyclic peptides corresponding to the 278-287 ((278)LLEDPVGTVA(287)) sequence of glycoprotein D (gD-1) of herpes simplex virus were synthesized for the analysis of the effect of cyclization on protection against enzymatic degradation. In this design, the turn-forming motif ((281)DPVG(284)) was positioned in the central part of the peptide and elongated by three amino acids at both termini. Cyclopeptide formation was achieved by the introduction of a peptide bond, a disulfide bridge or a thioether link. The stability of these peptides was compared in human serum and also in rat lysosomal preparations. The data obtained in 10% and 50% human serum show that all three types of cyclization enhanced the stability, but at different levels. Complete stability was only achieved by the introduction of a thioether link, while the presence of a disulfide or peptide bond resulted in improved, but partial resistance against hydrolytic decomposition. In lysosomal preparations the presence of cyclic primary structure provided full protection against enzymatic hydrolysis. Taken together, these findings indicate that by appropriate structural modification it is feasible to construct a synthetic antigen with high stability against enzymatic degradation in complex biological fluids. Further studies are in progress to identify enzymes responsible for degradation in diluted human sera as well as in the lysosomal preparations and to gain more detailed information on the mechanism of action.

Animals↗

PubMed2005

A new class of semisynthetic anthracycline glycoside antibiotics incorporating a squaric acid moiety.

Treatment of the squaric acid amide esters (7, 9) of anthracycline glycoside antibiotics with aliphatic and aromatic primary and secondary amines, amino acids, peptides and aminodeoxy sugars furnished the new asymmetric diamides 16-19, 25-30, 32, 34 and 38-40 in stereoselective reactions which do not require protecting group-manipulations. The IC50 = 0.12 microM value measured for daunorubicin (1) on human leukemia (HL-60) cells is comparable to those obtained for the daunomycin-L-leucyl squaric acid diamide (30, IC50 = 0.18 microM) and the corresponding D-galactosamine derivative (40, IC50=0.22 microM).

Anthracyclines↗

PubMed2005

Synthesis of linear, branched, and cyclic peptide chimera.

Chimeric peptides are unnatural constructs consisting of bioactive compounds from at least two different peptide(s) and/or protein(s) or two sequences from different parts of the same protein. Such multifunctional peptide combinations are prepared to enhance the biological activity or selectivity of their components. New biological effects can also be achieved with the chimera. In this chapter the synthesis of three different types of chimeric peptides will be described. In a linear chimera, two peptide epitopes from different parts of glycoprotein D (gD) of herpes simplex virus (HSV) are combined. A branched chimera, built from linear peptides, consists of tuftsin oligomers with immunostimulatory activity and an epitope peptide of HSV gD. The third compound is a cyclic chimeric molecule, where alpha-conotoxin GI as a host peptide is modified by the incorporation of a core epitope from HSV gD as a guest sequence.

Amino Acid Sequence↗

PubMed2005

Synthesis of peptide bioconjugates.

Bioconjugates play an important role in several fields of biomolecular and biomedicinal sciences. Protein/polypeptide-based conjugates with covalently attached epitope peptides are considered as potential synthetic vaccine candidates and/or target antigens in affinity-based bioassays. This chapter describes the synthesis of two- and three-component bioconjugates using water-soluble branched chain polymeric polypeptides with multiple amino and/or carboxyl groups as macromolecular partners and oligopeptides as epitopes with small molecular mass. The synthetic procedures outline three major strategies for the incorporation of multiple copies of uniformly oriented peptide epitopes. In the first example, chloroacetylated polypeptide is conjugated with SH-peptide to form a thioether linkage. Second, two independent oligopeptides are introduced into a macromolecule by amide and disulfide bonds, respectively. In the third example, a new procedure is reported for the formation of disulfide bridges by the use of Npys-modified polypeptide and SH-peptide.

Amino Acid Sequence↗

PubMed2004

Enzymic characterization with progress curve analysis of a collagen peptidase from an enthomopathogenic bacterium, Photorhabdus luminescens.

A proteolytic enzyme, Php-B ( Photorhabdus protease B), was purified from the entomopathogenic bacterium, Photorhabdus luminescens. The enzyme is intracellular, and its molecular mass is 74 kDa. Tested on various peptide and oligopeptide substrates, Php-B hydrolysed only oligopeptides, with significant activity against bradykinin and a 2-furylacryloyl-blocked peptide, Fua-LGPA (2-furylacryloyl-Leu-Gly-Pro-Ala; kcat=3.6x10(2) s(-1), K(m)=5.8x10(-5) M(-1), pH optimum approx. 7.0). The p K(a1) and the p K(a2) values of the enzyme activity (6.1 and 7.9 respectively), as well as experiments with enzyme inhibitors and bivalent metal ions, suggest that the activity of Php-B is dependent on histidine and cysteine residues, but not on serine residues, and that it is a metalloprotease, which most probably uses Zn2+ as a catalytic ion. The enzyme's ability to cleave oligopeptides that contain a sequence similar to collagen repeat (-Pro-Xaa-Gly-), bradykinin and Fua-LGPA (a synthetic substrate for bacterial collagenases and collagen peptidases), but not native collagens (types I and IV) or denatured collagen (gelatin), indicates that Php-B is probably a collagen peptidase, the first enzyme of this type to be identified in an insect pathogen, that might have a role in the nutrition of P. luminescens by degrading small collagen fragments. For the determination of enzyme kinetic constants, we fitted a numerically integrated Michaelis-Menten model to the experimental progress curves. Since this approach has not been used before in the characterization of proteases that are specific for the P1'-P4' substrate sites (e.g. collagenolytic enzymes), we present a comparison of this method with more conventional ones. The results confirm the reliability of the numerical integration method in the kinetic analysis of collagen-peptide-hydrolysing enzymes.

Bradykinin↗

PubMed2004

Synthesis, conformation, and immunoreactivity of new carrier molecules based on repeated tuftsin-like sequence.

Sequential oligopeptides based on a pentapeptide (TKPKG) derived from tuftsin with different lengths were synthesized by stepwise solid phase methodology. These highly soluble oligomers were nontoxic on mouse spleen cells, and other biological data suggested that tuftsin-like properties were also presented. The (TKPKG)n (n=2,4,6,8) oligopeptides were not immunogenic; however, they increased sheep red blood cells (SRBC) antigen specific antibody response in mice, demonstrating their immunostimulatory effect. Chemotactic activity was also found on J774 monocyte cells, while MRC5 fibroblasts were chemotactically nonresponders to the tested forms of tuftsin. These oligomers showed unordered and flexible structure by CD measurements, confirmed by computer modeling studies indicating also a fairly good accessibility of the epsilon-amino group of each lysine residue. Data suggest that these new oligotuftsin derivatives can be considered as promising carriers for synthetic vaccine.

Amino Acid Sequence↗