Two isomers of cis-aconytil-daunomycin (cAD) were isolated after the reaction of daunomycin with ... more Two isomers of cis-aconytil-daunomycin (cAD) were isolated after the reaction of daunomycin with cis-aconitic-anhydride. The structure of the isomers was identified by MS-spectroscopy and 1 H and 13 C NMR experiments. In contrast with the assumptions described earlier, our results show that the two isomers belong to the cis-and trans-isomers of the a-monoamide of cis-aconityldaunomycin, respectively. We found that the pH dependent daunomycin release is different for the two isomers. Comparative analysis of the in vitro antitumour effect of the isomers on c26 colon carcinoma and on MDA-MB 435P human breast carcinoma cell lines showed that cAD-1 is more potent than cAD-2, but the extent of differences is tumour cell dependent. The results of this study might be appreciated in the light of the use of acid-labile spacer for the design and preparation of protein/peptide conjugates of drugs by indicating that isomers could possess markedly different biological activity.
This report provides a detailed analysis on the influence of phosholipid bilayers on the conforma... more This report provides a detailed analysis on the influence of phosholipid bilayers on the conformation of poly[Lys(X(i)-DL-Ala(m))] (XAK, where X = Ser, Orn, Glu, or AcGlu) type branched polypeptides and their peptide conjugates. CD spectra of polycationic (SAK, OAK), amphoteric (EAK), or polyanionic (Ac-EAK) polylysine derivatives were recorded in 0.25M acetate buffer at pH 7.4 as well as in the presence of DPPC or DPPC/PG (95/5, 80/20 mol/mol) liposomes. Based on these data, two groups of polypeptides are described. Group one contains polypeptides with significantly ordered conformation even in buffer solution (SAK, AcEAK), which is essentially not altered by phospholipids. Group two, branched polypeptides (OAK, EAK), with only partially ordered conformation in aqueous solution in the presence of phospholipid bilayers with high PG content, could adopt more (EAK) or less (OAK) ordered alpha-helical structure depending on their charge properties. In addition, we report on the synthesis of two new sets of oligopeptide-branched polypeptide conjugates. Studies with selected conjugates suggest that these compounds are highly ordered in buffer solution almost regardless from the helix-forming ability of the carrier (AK, SAK, EAK) and from the hydrophilic/hydrophobic character of peptides attached (AVKDEL vs FWRGDLVFDFQV). Addition of phospholipid bilayers with different composition essentially had no modifying effect on conformation of conjugates. From this we can conclude that the covalently coupled oligopeptides has a predominant effect of the conformational properties of conjugates.
Calpains are intracellular cysteine proteases with several important physiological functions. Cal... more Calpains are intracellular cysteine proteases with several important physiological functions. Calpain inhibitors may be promising tools in the analysis of the function of the enzyme in diseases caused by overexpression/activation. Here, we report on the synthesis, solution conformation, and characterization of novel group of azapeptides whose sequences originate from an efficient m-calpain substrate, TPLKSPPPSPR, described by us earlier and possess varying levels of calpain inhibition. The Lys residue at P1 position was replaced with azaglycine (NH2 -NH-COOH) and further changes were made as follows: the N-terminal or/and C-terminal were truncated, amino acids were also changed at P3, P2, P'1, or P'2 positions. Our results indicate that the identity of amino acid moieties between P4 and P'5 positions is essential for the inhibitory activity. Only changes at position P3 (Pro) are tolerated. Azapeptide analogs, described in this communication could be considered as useful set of compounds for elucidation of the enzyme interaction at P and P' sites.
In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence ... more In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.
The results of conformational analysis of linear and cyclic peptides from the SALLEDPVG sequence ... more The results of conformational analysis of linear and cyclic peptides from the SALLEDPVG sequence of 276 284 glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable b-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH confirm presence of a type I b-turn 2 structure which includes the DPVG epitope core. ᮊ
Trends in Colloid and Interface Science XXIV, 2011
ABSTRACT Tuberculosis is a major problem throughout the world causing 1.9 million deaths annually... more ABSTRACT Tuberculosis is a major problem throughout the world causing 1.9 million deaths annually, and induces a major global public health problem. The pathogen responsible for the disease is Mycobacterium tuberculosis. Drug candidates expected to be specific inhibitors of dUTPase an essential enzyme of Mycobacterium tuberculosis were identified in silico. A phospholipid Langmuir monolayer formed at the liquid/air interface as a simple but versatile model of the cell membrane was applied to assess the membrane affinity of the drug candidates. The interaction of three different potential drug molecules TB501, TB502, and TB505 with lipid monolayer was characterised by tensiometry, and atomic force microscopy at different temperatures. The degree of penetration of drug candidates into the lipid film and the structural variation of drug penetrated lipid films revealed by atomic force microscopic images were compared.
Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known ... more Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known murine T cell epitope, both in T-B and B-T orientations, show that induction of high murine anti-MUC1 antibody titers is dependent on the presence and orientation of the T cell determinant. However, the sequential order of the epitopes does not affect binding of anti-B cell epitope antibodies to the constructs. Haplotype mismatching leads to a significant lowering of the anti-MUC1 antibody responses, implicating a central role for the T cell epitope in eliciting anti-B cell epitope responses. Secondary structure analysis by circular dichroism spectroscopy reveals the T-B construct to be partially ordered, while the B-T peptide adopts a highly ordered conformation in trifluoroethanol. These studies suggest that the sequential order of epitopes may significantly alter the immunogenicity of the peptide but may not necessarily affect its antigenicity. Immunogenicity of the peptide constructs...
Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofurano... more Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofuranosyl cytosine (ara-C) to human and bovine serum albumin (HSA, BSA) and to chemically modified albumin. The binding of 4-phenylbutyric acid to HSA was studied, too. Binding data were presented as Scatchard plots. There are two types of binding sites of different affinity for ara-C both on HSA and BSA. The relatively small value of affinity constant indicates that the pharmacological properties of ara-C might not be influenced very strongly by the HSA interaction or by competitive binding of other drugs. Selective chemical modifications of HSA with diethylpyrocarbonate (DEP) or o-nitrophenylsulfenyl chloride (NPS-Cl) reduce significantly the affinity of the strong binding area. On the other hand, the attachment of poly-alpha-L-glutamyl or poly-DL-alanyl side-chains to BSA increase the number of the strong and secondary binding sites and also the affinity at the first group of sites. Experim...
MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of... more MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were cha...
Selective delivery of antiparasitic or antibacterial drugs into infected macrophages could be a p... more Selective delivery of antiparasitic or antibacterial drugs into infected macrophages could be a promising approach for improved therapies. Methotrexate conjugate with branched chain polypeptides exhibited pronounced anti-Leishmania activity in vitro and in vivo as reported here earlier. To identify structural requirements for efficient uptake of branched polypeptides, we have studied murine bone marrow culture-derived macrophages (BMMphi) from 129/ICR mice. We report on the translocation characteristics of structurally closely related compounds labeled with 5(6)-carboxyfluorescein. We found that this process is dependent on experimental conditions (e.g. polypeptide concentration, incubation time, and temperature). Using specific inhibitors as well as macrophages from wild-type and class-A scavenger receptor knockout (SR-A -/-) mice, we demonstrated that SR-A was involved in the endocytosis of some polypeptides depending on their charge. Uptake could be blocked by unlabeled polypepti...
Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Al... more Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala(m))] (AK), poly[Lys(Ser(i)-DL-Ala(m))] (SAK), poly[Lys(DL-Ala(m)-Leu(i))] (ALK)), or amphoteric (poly[Lys(Glu(i)-DL-Ala(m))] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mi...
ABSTRACT A new group of synthetic macromolecular conjugates was synthesised in which poly(Lys) or... more ABSTRACT A new group of synthetic macromolecular conjugates was synthesised in which poly(Lys) or a branched polypeptide poly[Lys-(DL-Alam)] (m≅3) were used as carrier and 4-(ethoxymethylene)-2–phenyl–5 (4H)-oxazolone as hapten. These conjugates were characterized by amino acid analysis, identification of terminal residues of the side chain, sedimentation analysis. The conformation of conjugates and of carrier polypeptides were analyzed by circular dichroism spectroscopy in water solutions at various pH and ionic strengths. These data indicated a marked dependence of the conformation of the conjugate on the structure of carrier and on the number of the side chain terminal haptens (phOx). In vitro cytotoxicity of these polymers was also investigated using two different assays, by measuring the viability of isolated rat liver cells and the effect on growth of HeLa cells. Toxicity of polypeptides could be diminished by conjugates containing a higher amount of oxazolone. These conjugates with defined conformation and toxicological properties are considered suitable to analyse the carrier function of branched polypeptides, particularly the interaction with the immunological network.
cells could play a role in cancer progression. When analysing the expression of BE in 30 human me... more cells could play a role in cancer progression. When analysing the expression of BE in 30 human melanoma biopsies obtained from patients, we found a correlation between beta endorphin expression and stage of the malignancy (p < 0, 05). We analysed the potential role of BE in preventing immune response against tumor cells and performed a mice model of cancer progression by subcutaneous injection of melanoma B16 cells to both mu opioid receptor deficient mice (MOR -/-) and their WT counterparts. A profound decrease in tumor growth was observed in MOR -/-mice compared to WT animals (median volume 0,2 cm3 versus 0,8 cm3 at day 15 post injection; p < 0,01). This was paralleled by a significant higher infiltration of CD4+, CD8+, NK and dendritic cells at tumor site of MOR -/mice determined by flow cytometry and immunohistochemistry. Adoptive transfer experiment with PKH-26-labeled MOR -/-leukocytes in combination with PKH-67-labeled WT leukocytes demonstrated that the higher presence of immune cells was not du to a higher recruitment of cells at tumor sites, but rather to proliferation and activation of leukocytes. NK cell activation was indeed increased by the use of BE-blocking antibody.
International journal of peptide and protein research, 1995
2-Phenyl-4-ethoxymethylene-5(4H)-oxazolone (PhOx = CHOEt) was reacted with methylamine, and 2-phe... more 2-Phenyl-4-ethoxymethylene-5(4H)-oxazolone (PhOx = CHOEt) was reacted with methylamine, and 2-phenyloxazole-4-carboxylic acid was coupled with methylamine. The spectroscopic properties of the two products were compared in order to confirm that aminolysis of PhOx = CHOEt occurs by displacement of the ethoxy group to give 2-phenyl-4-(substituted-methylene)-5(4H)-oxazolones and not by attack at the oxazolone-carbonyl followed by rearrangement to give 2-phenyloxazole-4-carboxamides. Ten crystalline conjugates were prepared and characterized by reacting PhOx = CHOEt with an excess of unprotected di- and trifunctional amino acid anions followed by purification by washing them with hydrochloric acid.
Calpains are intracellular cysteine proteases with important physiological functions. Up- or down... more Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.
The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotei... more The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.
A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximat... more A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceed...
The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex ... more The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography -mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.
Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an ... more Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca 2+ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α-helical conformation in 50% CD 3 OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca 2+ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain.
Two isomers of cis-aconytil-daunomycin (cAD) were isolated after the reaction of daunomycin with ... more Two isomers of cis-aconytil-daunomycin (cAD) were isolated after the reaction of daunomycin with cis-aconitic-anhydride. The structure of the isomers was identified by MS-spectroscopy and 1 H and 13 C NMR experiments. In contrast with the assumptions described earlier, our results show that the two isomers belong to the cis-and trans-isomers of the a-monoamide of cis-aconityldaunomycin, respectively. We found that the pH dependent daunomycin release is different for the two isomers. Comparative analysis of the in vitro antitumour effect of the isomers on c26 colon carcinoma and on MDA-MB 435P human breast carcinoma cell lines showed that cAD-1 is more potent than cAD-2, but the extent of differences is tumour cell dependent. The results of this study might be appreciated in the light of the use of acid-labile spacer for the design and preparation of protein/peptide conjugates of drugs by indicating that isomers could possess markedly different biological activity.
This report provides a detailed analysis on the influence of phosholipid bilayers on the conforma... more This report provides a detailed analysis on the influence of phosholipid bilayers on the conformation of poly[Lys(X(i)-DL-Ala(m))] (XAK, where X = Ser, Orn, Glu, or AcGlu) type branched polypeptides and their peptide conjugates. CD spectra of polycationic (SAK, OAK), amphoteric (EAK), or polyanionic (Ac-EAK) polylysine derivatives were recorded in 0.25M acetate buffer at pH 7.4 as well as in the presence of DPPC or DPPC/PG (95/5, 80/20 mol/mol) liposomes. Based on these data, two groups of polypeptides are described. Group one contains polypeptides with significantly ordered conformation even in buffer solution (SAK, AcEAK), which is essentially not altered by phospholipids. Group two, branched polypeptides (OAK, EAK), with only partially ordered conformation in aqueous solution in the presence of phospholipid bilayers with high PG content, could adopt more (EAK) or less (OAK) ordered alpha-helical structure depending on their charge properties. In addition, we report on the synthesis of two new sets of oligopeptide-branched polypeptide conjugates. Studies with selected conjugates suggest that these compounds are highly ordered in buffer solution almost regardless from the helix-forming ability of the carrier (AK, SAK, EAK) and from the hydrophilic/hydrophobic character of peptides attached (AVKDEL vs FWRGDLVFDFQV). Addition of phospholipid bilayers with different composition essentially had no modifying effect on conformation of conjugates. From this we can conclude that the covalently coupled oligopeptides has a predominant effect of the conformational properties of conjugates.
Calpains are intracellular cysteine proteases with several important physiological functions. Cal... more Calpains are intracellular cysteine proteases with several important physiological functions. Calpain inhibitors may be promising tools in the analysis of the function of the enzyme in diseases caused by overexpression/activation. Here, we report on the synthesis, solution conformation, and characterization of novel group of azapeptides whose sequences originate from an efficient m-calpain substrate, TPLKSPPPSPR, described by us earlier and possess varying levels of calpain inhibition. The Lys residue at P1 position was replaced with azaglycine (NH2 -NH-COOH) and further changes were made as follows: the N-terminal or/and C-terminal were truncated, amino acids were also changed at P3, P2, P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;1, or P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;2 positions. Our results indicate that the identity of amino acid moieties between P4 and P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;5 positions is essential for the inhibitory activity. Only changes at position P3 (Pro) are tolerated. Azapeptide analogs, described in this communication could be considered as useful set of compounds for elucidation of the enzyme interaction at P and P&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; sites.
In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence ... more In the altered form of MUC1 mucin associated with breast cancer, the highly immunogenic sequence PDTRPAP is exposed, and may be an immunologically relevant target for the development of diagnostics or cancer immunotherapy. In this study, we report the preparation and antibody binding properties of monomeric and dimeric MUC1 peptides containing the epitope region recognized by monoclonal antibody (mAb) C595. Peptides contained a single or two copies of the whole 20-mer repeat unit (VTSAPDTRPAPGSTAPPAHG) of MUC1 protein. MUC1 40-mer peptides were prepared by the condensation of semi-protected fragments of the repeat unit, in solution or by chemical ligation. In the first case, cyclohexyl-type protecting groups were used for the synthesis of semi-protected fragments by the Boc/Bzl strategy. Unprotected fragments were used in the chemical ligation to produce thioether linkages. In one of the fragments, a Gly residue was replaced by Cys at the C-terminus and the other fragment was chloroacetylated at the N-terminus. In addition, the short peptide APDTRPAPG, and its disulfide dimer, (APDTRPAPGC)(2) were produced. The antibody binding properties of these MUC1 peptide constructs were tested by competition enzyme-linked immunosorbent assay (ELISA). The short epitope region peptide, APDTRPAPG and its dimer (APDTRPAPGC)(2) showed higher IC(50) values (IC(50) = 56.3 and 53.2 micromol/l, respectively). While the 20-mer peptide (IC(50) = 25.9 micromol/l) and more markedly its 40-mer dimers (IC(50) = 0.62 and 0.78 micromol/l) were recognized better. CD data obtained in water or in TFE indicated no significant conformational differences between the 20-mer and 40-mer peptides. We found a high level of similarity between the binding properties of the 40-mer peptides with amide or thioether links, providing a new possibility to build up oligomeric MUC1 peptides by thioether bond formation.
The results of conformational analysis of linear and cyclic peptides from the SALLEDPVG sequence ... more The results of conformational analysis of linear and cyclic peptides from the SALLEDPVG sequence of 276 284 glycoprotein D of Herpes simplex virus are presented. The epitope peptides were synthesized by SPPS and on resin cyclization was applied for preparation of cyclic compounds. Circular dichroism spectroscopy, Fourier-transform infrared spectroscopy and nuclear magnetic resonance (NMR) were used to determine of the solution structure of both linear and cyclic peptides. The results indicated that the cyclopeptides containing the core of the epitope (DPVG) as a part of the cycle have more stable b-turn structure than the linear peptides or the cyclic analogues, where the core motif is not a part of the cycle. NMR study of H-SALLc(EDPVGK)-NH confirm presence of a type I b-turn 2 structure which includes the DPVG epitope core. ᮊ
Trends in Colloid and Interface Science XXIV, 2011
ABSTRACT Tuberculosis is a major problem throughout the world causing 1.9 million deaths annually... more ABSTRACT Tuberculosis is a major problem throughout the world causing 1.9 million deaths annually, and induces a major global public health problem. The pathogen responsible for the disease is Mycobacterium tuberculosis. Drug candidates expected to be specific inhibitors of dUTPase an essential enzyme of Mycobacterium tuberculosis were identified in silico. A phospholipid Langmuir monolayer formed at the liquid/air interface as a simple but versatile model of the cell membrane was applied to assess the membrane affinity of the drug candidates. The interaction of three different potential drug molecules TB501, TB502, and TB505 with lipid monolayer was characterised by tensiometry, and atomic force microscopy at different temperatures. The degree of penetration of drug candidates into the lipid film and the structural variation of drug penetrated lipid films revealed by atomic force microscopic images were compared.
Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known ... more Synthetic peptide constructs, co-linearly linking a MUC1 mucin B cell epitope peptide to a known murine T cell epitope, both in T-B and B-T orientations, show that induction of high murine anti-MUC1 antibody titers is dependent on the presence and orientation of the T cell determinant. However, the sequential order of the epitopes does not affect binding of anti-B cell epitope antibodies to the constructs. Haplotype mismatching leads to a significant lowering of the anti-MUC1 antibody responses, implicating a central role for the T cell epitope in eliciting anti-B cell epitope responses. Secondary structure analysis by circular dichroism spectroscopy reveals the T-B construct to be partially ordered, while the B-T peptide adopts a highly ordered conformation in trifluoroethanol. These studies suggest that the sequential order of epitopes may significantly alter the immunogenicity of the peptide but may not necessarily affect its antigenicity. Immunogenicity of the peptide constructs...
Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofurano... more Equilibrium dialysis measurements were carried out to study the binding of 1-beta-D-arabinofuranosyl cytosine (ara-C) to human and bovine serum albumin (HSA, BSA) and to chemically modified albumin. The binding of 4-phenylbutyric acid to HSA was studied, too. Binding data were presented as Scatchard plots. There are two types of binding sites of different affinity for ara-C both on HSA and BSA. The relatively small value of affinity constant indicates that the pharmacological properties of ara-C might not be influenced very strongly by the HSA interaction or by competitive binding of other drugs. Selective chemical modifications of HSA with diethylpyrocarbonate (DEP) or o-nitrophenylsulfenyl chloride (NPS-Cl) reduce significantly the affinity of the strong binding area. On the other hand, the attachment of poly-alpha-L-glutamyl or poly-DL-alanyl side-chains to BSA increase the number of the strong and secondary binding sites and also the affinity at the first group of sites. Experim...
MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of... more MUC2 glycoprotein, produced by the epithelium of the colon and built up mainly of repeat units of (1) PTTTPITTTTTVTPTPTPTGTQT(23) , can be overexpressed or underglycosylated in gastrointestinal diseases, e.g. in case of colon carcinoma. We have been studying the epitope structure of the MUC2 by focusing on the repeat unit with the mucin peptide specific MAb 996 monoclonal antibody. This antibody recognizes the (18) PTGTQ(22) sequence as minimal, and (16) PTPTGTQ(22) as optimal epitope within the underglycosylated glycoprotein. In this article, we aim to clarify the effect of glycosylation of the epitope on MAb 996 antibody binding including its correlation with the secondary structure of the modified peptides: glycosylation in the epitope core and in the flank. For this we have prepared the (16) PTPTGTQ(22) peptide glycosylated with N-acetylgalactoseamine (Tn antigen) in position 17, 19, 21, or on all three threonines. The MAb 996 antibody binding properties of the peptides were cha...
Selective delivery of antiparasitic or antibacterial drugs into infected macrophages could be a p... more Selective delivery of antiparasitic or antibacterial drugs into infected macrophages could be a promising approach for improved therapies. Methotrexate conjugate with branched chain polypeptides exhibited pronounced anti-Leishmania activity in vitro and in vivo as reported here earlier. To identify structural requirements for efficient uptake of branched polypeptides, we have studied murine bone marrow culture-derived macrophages (BMMphi) from 129/ICR mice. We report on the translocation characteristics of structurally closely related compounds labeled with 5(6)-carboxyfluorescein. We found that this process is dependent on experimental conditions (e.g. polypeptide concentration, incubation time, and temperature). Using specific inhibitors as well as macrophages from wild-type and class-A scavenger receptor knockout (SR-A -/-) mice, we demonstrated that SR-A was involved in the endocytosis of some polypeptides depending on their charge. Uptake could be blocked by unlabeled polypepti...
Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Al... more Methotrexate (MTX) has been coupled to various structurally related, polycationic (poly[Lys(DL-Ala(m))] (AK), poly[Lys(Ser(i)-DL-Ala(m))] (SAK), poly[Lys(DL-Ala(m)-Leu(i))] (ALK)), or amphoteric (poly[Lys(Glu(i)-DL-Ala(m))] (EAK)) synthetic branched polypeptides containing poly[L-Lys] backbone by the aid of BOP reagent. The average degree of MTX incorporation was found to be dependent on the charge properties of the polymer. Under the experimental conditions used, the molar substitution ratio achieved was higher for polycations (25%) than for the amphoteric polypeptide (10%). We have studied the effect of polycationic polypeptides on Leishmania donovani infection. Results demonstrated that MTX conjugates in which the drug is covalently attached to carrier have pronounced leishmanicid activity. In this communication we showed that (a) a branched polypeptide-methotrexate conjugate with a polycationic carrier (ALK) increases the effect of MTX against Leishmania donovani infection in mi...
ABSTRACT A new group of synthetic macromolecular conjugates was synthesised in which poly(Lys) or... more ABSTRACT A new group of synthetic macromolecular conjugates was synthesised in which poly(Lys) or a branched polypeptide poly[Lys-(DL-Alam)] (m≅3) were used as carrier and 4-(ethoxymethylene)-2–phenyl–5 (4H)-oxazolone as hapten. These conjugates were characterized by amino acid analysis, identification of terminal residues of the side chain, sedimentation analysis. The conformation of conjugates and of carrier polypeptides were analyzed by circular dichroism spectroscopy in water solutions at various pH and ionic strengths. These data indicated a marked dependence of the conformation of the conjugate on the structure of carrier and on the number of the side chain terminal haptens (phOx). In vitro cytotoxicity of these polymers was also investigated using two different assays, by measuring the viability of isolated rat liver cells and the effect on growth of HeLa cells. Toxicity of polypeptides could be diminished by conjugates containing a higher amount of oxazolone. These conjugates with defined conformation and toxicological properties are considered suitable to analyse the carrier function of branched polypeptides, particularly the interaction with the immunological network.
cells could play a role in cancer progression. When analysing the expression of BE in 30 human me... more cells could play a role in cancer progression. When analysing the expression of BE in 30 human melanoma biopsies obtained from patients, we found a correlation between beta endorphin expression and stage of the malignancy (p < 0, 05). We analysed the potential role of BE in preventing immune response against tumor cells and performed a mice model of cancer progression by subcutaneous injection of melanoma B16 cells to both mu opioid receptor deficient mice (MOR -/-) and their WT counterparts. A profound decrease in tumor growth was observed in MOR -/-mice compared to WT animals (median volume 0,2 cm3 versus 0,8 cm3 at day 15 post injection; p < 0,01). This was paralleled by a significant higher infiltration of CD4+, CD8+, NK and dendritic cells at tumor site of MOR -/mice determined by flow cytometry and immunohistochemistry. Adoptive transfer experiment with PKH-26-labeled MOR -/-leukocytes in combination with PKH-67-labeled WT leukocytes demonstrated that the higher presence of immune cells was not du to a higher recruitment of cells at tumor sites, but rather to proliferation and activation of leukocytes. NK cell activation was indeed increased by the use of BE-blocking antibody.
International journal of peptide and protein research, 1995
2-Phenyl-4-ethoxymethylene-5(4H)-oxazolone (PhOx = CHOEt) was reacted with methylamine, and 2-phe... more 2-Phenyl-4-ethoxymethylene-5(4H)-oxazolone (PhOx = CHOEt) was reacted with methylamine, and 2-phenyloxazole-4-carboxylic acid was coupled with methylamine. The spectroscopic properties of the two products were compared in order to confirm that aminolysis of PhOx = CHOEt occurs by displacement of the ethoxy group to give 2-phenyl-4-(substituted-methylene)-5(4H)-oxazolones and not by attack at the oxazolone-carbonyl followed by rearrangement to give 2-phenyloxazole-4-carboxamides. Ten crystalline conjugates were prepared and characterized by reacting PhOx = CHOEt with an excess of unprotected di- and trifunctional amino acid anions followed by purification by washing them with hydrochloric acid.
Calpains are intracellular cysteine proteases with important physiological functions. Up- or down... more Calpains are intracellular cysteine proteases with important physiological functions. Up- or downregulation of their expression can be responsible for several diseases, therefore specific calpain inhibitors may be considered as promising candidates for drug discovery. In this paper we describe the synthesis and characterization of a new class of inhibitors derived from the analysis of amino acid preferences in primed and unprimed sites of calpains by incorporation of l- or d-epoxysuccinyl group (Eps). Amino acids for replacement were chosen by considering the substrate preference of calpain 1 and 2 enzymes. The compounds were characterized by RP-HPLC, amino acid analysis and ESI-MS. Selectivity of the compounds was studied by using calpain 1 and 2; and cathepsin B. We have identified five calpain specific inhibitors with different extent of selectivity. Two of these also exhibited isoform selectivity. Compound NH2-Thr-Pro-Leu-(d-Eps)-Thr-Pro-Pro-Pro-Ser-NH2 proved to be a calpain 2 enzyme inhibitor with at least 11.8-fold selectivity, while compound NH2-Thr-Pro-Leu-(l-Eps)-Ser-Pro-Pro-Pro-Ser-NH2 possesses calpain 1 enzyme inhibition with at least 4-fold selectivity. The results of molecular modeling calculations suggest that the orientation of the bound inhibitor in the substrate binding cleft is markedly dependent on the stereochemistry of the epoxysuccinyl group.
The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotei... more The fine structure of the antigenic determinants of herpes simplex virus type 1 and 2 glycoprotein D (gD) was analyzed to determine whether structural differences underlie the differential immunogenicity of these glycoproteins. A region common to herpes simplex virus type 1 and 2 gD (amino acid residues 11 to 19) and two sites specific for herpes simplex virus type 2 gD (one determined by proline at position 7, the other determined by asparagine at position 21) were localized within the N-terminal 23 amino acids of gD by synthesis of peptides and comparison of their cross-reactivity with antisera raised to herpes simplex virus type 1 and 2 gD. The secondary structure of these peptides, as predicted by computer analysis, is discussed in relation to their immunogenicity.
A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximat... more A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceed...
The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex ... more The mass spectrometric analysis of the immunodominant epitope region (273-284) of herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) showed a favoured fission at the Asp-Pro peptide bond. The fast atom bombardment collision induced dissociation (FAB-CID) study of closely related X-Pro peptides documented that neither the length nor the amino acid composition of the peptide has a significant influence on this preferential cleavage. At the same time the DP bond proved to be sensitive to acidic conditions in the course of peptide synthesis. These observations prompted us to compare the chemical and mass spectrometric stability of a new set of nonapeptides related to the 273-284 epitope region of gD, i.e. SALLEDPVG and SALLEXPVG peptides, where X = A, K, I, S, F, E or D, respectively. The chemical stability of these peptides during acidic hydrolysis was investigated by electrospray ionization mass spectrometry (ESI-MS) and the products were identified by ESI-MS and on-line high performance liquid chromatography -mass spectrometry (HPLC-MS). The mass spectrometric fragmentation and bond stability of the untreated peptide samples were also studied using ESI-MS and liquid secondary ion mass spectrometry (LSIMS). Both the chemical hydrolysis and the mass spectrometric fragmentation showed that the Asp-Pro bond could easily be cleaved, while the KP bond proved to be stable under both circumstances. On the other hand, the XP bond (X = A, I, S, F or E) fragmented easily under the mass spectrometric conditions, but was not sensitive to the acidolysis.
Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an ... more Calpastatin, the endogenous inhibitor of calpain, a cysteine protease in eukaryotic cells, is an intrinsically unstructured protein, which upon binding to the enzyme goes through a conformational change. Peptides calpA (SGKSGMDAALDDLIDTLGG) and calpC (SKPIGPDDAIDALSSDFTS), corresponding to the two conserved subdomains of calpastatin, are known to activate calpain and increase the Ca 2+ sensitivity of the enzyme. Using solution NMR spectroscopy, here we show that calpA and calpC are disordered in water but assume an α-helical conformation in 50% CD 3 OH. The position and length of the helices are in agreement with those described in the literature for the bound state of the corresponding segments of calpastatin suggesting that the latter might be structurally primed for the interaction with its target. According to our data, the presence of Ca 2+ induces a backbone rearrangement in the peptides, an effect that may contribute to setting the fine conformational balance required for the interaction of the peptides with calpain.
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Papers by Ferenc Hudecz