Biomedical subjects

F Hudecz

Publications and source records attributed to F Hudecz.

79 records · Page 5Linked to original sources

PubMedDate not supplied

Synthesis, conformation, biodistribution, and hormone-related in vitro antitumor activity of a gonadotropin-releasing hormone antagonist-branched polypeptide conjugate.

Since permanently high levels of GnRH analogues are necessary to exert direct and/or indirect antitumor effect on mammary tumors, much emphasis was put on the development of retarded-release devices (e.g. microcapsules) for GnRH derivatives. Alternatively, these compounds can be covalently coupled to high-molecular mass carrier molecules for the design of bioconjugates acting as (a) prodrugs producing prolonged release or (b) macromolecular therapeutics. In order to evaluate the feasibility of this approach, a prototype construct has been prepared with a potent GnRH antagonist Ac(D-Trp1,3, D-Cpa2, D-Lys6, D-Ala10)-GnRH (MI-1544). As a carrier, a representative of a new generation of synthetic, biodegradable branched poly[Lys(Xi-DL-Alam)] (XAK) type polypeptides with poly(L-lysine) backbone has been used in which X is an acetylated derivative of glutamic acid (AcEAK). This polyanionic polypeptide with free gamma-carboxyl groups was conjugated to MI-1544, which has only a single amino group at position 6. In this paper, we describe (i) the synthesis and structure (primary structure, conformation) properties of the MI-1544-AcEAK conjugate with a 33% degree of substitution, (ii) the effect of the covalent attachment of MI-1544 to AcEAK on its blood clearance and tissue distribution, and (iii) the hormone-related indirect (ovulation inhibitory) or direct (antiproliferative) antitumor activity of the conjugate studied by in vitro assays. Data obtained with 111In- and 125I-labeled conjugates have demonstrated that in fact the body/blood survival of MI-1544 was prolonged by 1.5-3 times. The direct in vitro antitumor effect of MI-1544 was maintained or even enhanced in the MI-1544-AcEAK conjugate. Furthermore, we have shown that this conjugate was able to antagonize the effect of GnRH in vitro or to act as free MI-1544 both in short- and long-term inhibition of ovulation even after single subcutaneous injection. These data suggest that it is feasible to use a biodegradable polymeric polypeptide for development of a macromolecular therapeutic with GnRH antagonists.

Animals↗

PubMedDate not supplied

Synthesis and in vitro T-cell immunogenicity of conjugates with dual specificity: attachment of epitope peptides of 16 and 38 kDa proteins from Mycobacterium tuberculosis to branched polypeptide.

T-cell epitope containing peptides covalently attached to macromolecular carriers can be considered as synthetic immunogens for the development of skin-test diagnostics and of vaccines. As a carrier, an amphoteric branched chain polypeptide, poly[Lys-(Glui-DL-Alam)] (EAK) with poly(L-lysine) backbone has been used. This polypeptide with free alpha-amino and gamma-carboxyl groups at the end of the side chains was conjugated with peptides representing two immunodominant regions of the 16 and 38 kDa proteins of Mycobacterium tuberculosis, respectively. Peptide C91SEFAYGSFVRTVSLPVGADE110 was elongated by Cys at the N-terminal and attached to the carrier containing protected SH groups to form disulfide bridges. Peptide 65FNLWGPAFHERYPNVTITA83 was conjugated to the 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP) modified and acetylated EAK by introducing amide bond between the free alpha-amino group of peptide and the free gamma-COOH group of Glu at the terminal position of the branches. This strategy lead to chemically well-defined synthetic immunogens that contain two different epitopes in multiple copies covalently linked to a synthetic branched polypeptide carrier. In vitro T-cell immunogenicity of a prototype conjugate was studied using T-cell hybridomas, lymph node cells from 38 kDa protein immunized mice, and human peripheral blood mononuclear cell (PBMC) cultures from sensitized individuals. These data document that the specific T-cell stimulatory effect of each mycobacterial epitope was maintained in this conjugate. Taken together, these findings suggest that it is feasible to use a biodegradable polymeric polypeptide for producing macromolecular bioconjugates for the stimulation of T-cell responses.

Amino Acid Sequence↗

PubMedDate not supplied

Carrier design: new generation of polycationic branched polypeptides containing OH groups with prolonged blood survival and diminished in vitro cytotoxicity.

For the construction of macromolecule-drug conjugates, it is important to provide rational basis to the selection of proper carrier. With respect to the importance of the side-chain structure and charge of the branched polypeptides in biological properties, we have prepared a new class of branched polypeptides with single or multiple hydroxyl groups and studied their solution conformation, in vitro cytotoxicity, biodistribution, and immunoreactivity. For comparative studies, polypeptides were designed to contain serine at various positions of the side chains, varying also the number. Ser was attached to the end of oligo(DL-Ala) side chains grafted to polylysine resulting polypeptides with the general formula poly[Lys(Ser(i)-DL-Ala(m))], (SAK). Ser was also coupled directly to the polylysine backbone poly[Lys(Ser(i))] (S(i)K) and then elongated by polymerization of N-carboxy-DL-Ala anhydride resulting poly[Lys(DL-Ala(m)-Ser(i))] (ASK). An additional polymer was also prepared, but instead of the oligo(DL-Ala) branches, oligo(DL-Ser) side chains were introduced (poly[Lys(DL-Ser(m))], SK). The presence of hydroxyl groups resulted in compounds with improved of water solubility. CD spectra of polypeptides showed significant differences correlating with the position and numbers of Ser residues in the side chains. Under physiological conditions, polycationic polypeptides assumed ordered secondary structure (S(i)K and LSK) or partially unordered conformation (SK, SAK, and ASK). Data of selected polymers demonstrate that these polycationic compounds are essentially nontoxic in vitro on normal rat liver or mouse spleen cells and have no cytostatic effect on mouse colorectal carcinoma C26 cells. The blood clearance and biodistribution of these derivatives were greatly dependent on the position and number of Ser residues in the branches and possess a rather extended blood survival in mice. Polypeptides were taken up predominantly by the liver and kidney (S(i)K, LSK, and ASK) or kidney and lung (SK and SAK). The best survival in the blood was found with SAK, representing the first polycationic branched polypeptide, which show extended blood clearance. The relative position of Ser residue had also a marked influence on the immunogenicity of polypeptides. The characteristics of the antibody response to polypeptide containing Ser at the end of the branches (SAK) or adjacent to the polylysine backbone (ASK) was also dependent on the genetic background of the mouse strains. We also found that these compounds have no effect on to the SRBC-specific humoral immune response, indicating the lack of nonspecific immunostimulatory potential. In conclusion, these studies suggest that synthetic branched polypeptides with Ser can be considered as candidates for constructing suitable conjugates for drug/epitope delivery. It is not only due to the presence of hydroxyl group to be used for oxime chemistry but also to their beneficial biological features.

Adjuvants, Immunologic↗

PubMedDate not supplied

Phospholipid-model membrane interactions with branched polypeptide conjugates of a hepatitis A virus peptide epitope.

To establish correlation between structural properties (charge, composition, and conformation) and membrane penetration capability, the interaction of epitope peptide-carrier constructs with phospholipid model membranes was studied. For this we have conjugated a linear epitope peptide, (110)FWRGDLVFDFQV(121) (110-121), from VP3 capside protein of the Hepatitis A virus with polylysine-based branched polypeptides with different chemical characteristics. The epitope peptide elongated by one Cys residue at the N-terminal [C(110-121)] was attached to poly[Lys-(DL-Ala(m)()-X(i)())] (i < 1, m approximately 3), where x = ø(AK), Ser (SAK), or Glu (EAK) by the amide-thiol heterobifunctional reagent, 3-(2-pyridyldithio)propionic acid N-hydroxy-succinimide ester. The interaction of these polymer-[C(110-121)] conjugates with phospholipid monolayers and bilayers was studied using DPPC and DPPC/PG (95/5 mol/mol) mixture. Changes in the fluidity of liposomes induced by these conjugates were detected by using two fluorescent probes 1,6-diphenyl-1,3, 5-hexatriene (DPH) and sodium anilino naphthalene sulfonate (ANS). The binding of conjugates to the model membranes was compared and the contribution of the polymer component to these interactions were evaluated. We found that conjugates with polyanionic/EAK-[C(110-121)] or polycationic/SAK-[C(110-121)], AK-[C(110-121)]/character were capable to form monomolecular layers at the air/water interface with structure dependent stability in the following order: EAK-[C(110-121)] > SAK-[C(110-121)] > AK-[C(110-121)]. Data obtained from penetration studies into phospholipid monolayers indicated that conjugate insertion is more pronounced for EAK-[C(110-121)] than for AK-[C(110-121)] or SAK-[C(110-121)]. Changes in the fluorescence intensity and in polarization of fluorescent probes either at the polar surface (ANS) or within the hydrophobic core (DPH) of the DPPC/PG liposomes suggested that all three conjugates interact with the outer surface of the bilayer. Marked penetration was documented by a significant increase of the transition temperature only with the polyanionic compound/EAK-[C(110-121)]. Taken together, we found that the binding/penetration of conjugates to phospholipid model membranes is dependent on the charge properties of the constructs. Considering that the orientation and number of VP3 epitope peptides attached to branched polypeptides were almost identical, we can conclude that the structural characteristics (amino acid composition, charge, and surface activity) of the carrier have a pronounced effect on the conjugate-phospholipid membrane interaction. These observations suggest that the selection of polymer carrier for epitope attachment might significantly influence the membrane activity of the conjugate and provide guidelines for adequate presentation of immunogenic peptides to the cells.

1,2-Dipalmitoylphosphatidylcholine↗

PubMedDate not supplied

Conjugation of epitope peptides with SH group to branched chain polymeric polypeptides via Cys(Npys).

Since bioconjugates may play an important role as therapeutics in the future, the development of new and effective conjugation strategies is necessary. For the attachment of peptide-like molecules to carriers, there are two main coupling methods involving amide or disulfide bonds. Conjugation through an amide bond can be achieved in several well-defined ways known from peptide chemistry. However, the formation of disulfide bridges between cysteine-containing peptides and carrier molecules still has some problems. In this paper, we describe a novel approach in which the carrier polypeptide is modified by 3-nitro-2-pyridinesulfenyl (Npys)-protected cysteine and this derivative has been applied for conjugation of Cys-containing epitope peptides with poly(L-lysine)-based branched polypeptides. Considering the stability of Npys group in the presence of pentafluorophenol, Boc-Cys(Npys)-OPfp dervivative was selected for introduction to the N-terminal of branches of polypeptides backbone. The branches of the polymers were built up from oligo(DL-alanine) (poly[Lys(DL-Ala(m))], AK) and elongated by an optically active amino acid [poly[Lys(X(i)-DL-Ala(m))], XAK]. We found that the nature of X (Glu, Ser, Thr) has great influence on the incorporation of the protected cysteine residue. Herpes simplex virus and adenovirus epitope peptides were conjugated to Boc-Cys(Npys)-modified polypeptides. Results indicate that the incorporation of epitope peptides depends on the number of Npys group on the polymers as well as on the presence/absence of Boc-protecting group on the Cys residue. This new class of Cys(Npys)-derivatized branched polypeptides is stable for a couple of months and suitable for effective preparation of epitope peptide conjugates possessing increased water solubility.

Affinity Labels↗

PubMedDate not supplied

Epitope mapping of the 273-284 region of HSV glycoprotein D by synthetic branched polypeptide carrier conjugates.

To investigate the antigenicity of a predicted epitope region of herpes simplex virus gD, peptides comprising the 273-284 sequence have been synthesized and conjugated to a branched polypeptide with polylysine backbone (poly[L-Lys-(DL-Alam)], AK). In order to analyze the effect of the carrier on the solution conformation of the potential peptide-epitopes, three peptides (273-284, 273-281 and 276-284) and their polypeptide conjugates were studied by CD spectroscopy in PBS or in TFE. In immunized BALB/c and CBA mice, the level of peptide-, conjugate- and carrier-specific antibody responses were measured. Conjugates with synthetic polypeptide carrier AK induced epitope-specific IgG responses, accompanied by the appearance of a low level of carrier-specific antibodies. The cross-reactivity pattern of induced antibodies revealed the presence of at least two functionally distinct, overlapping epitopes, the availability of which was influenced by flanking residues at the N-terminus. Preimmunization of BALB/c or CBA mice with the [276-284]-AK conjugate resulted in the production of HSV-specific antibodies and in prolonged survival of animals infected with a lethal dose of herpes simplex virus. The degree of protection was comparable to that of [1-23]-AK conjugate (30).

Alanine↗

PubMedDate not supplied

Sequential order of T and B cell epitopes affects immunogenicity but not antibody recognition of the B cell epitope.

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 may be governed by subtle differences in secondary structure, leading to variation in the way peptides are presented or processed within cells governing immune responses. These findings have relevance for the construction of peptides to be utilized as potential synthetic vaccines and for the design of peptide immunogens.

Amino Acid Sequence↗