Peptide bioconjugates for intracellular targeting

The increasing number of papers in which bioconjugates of oligo- and/or polypeptides are present indicates the importance and potential of these constructs. In this contribution there is an attempt to provide an overview of our results concerning the preparation, functional characterization and mainly biomedicinal application of conjugates with chemotherapeutic agents against tumour, infectious diseases, with T-cell epitope peptides or with “reporter” entities to be used also for mechanistic studies prepared in our laboratories last decades

Pitfalls in the synthesis of fluorescent methotrexate oligopeptide conjugates

Abstract Methotrexate (MTX) conjugates with poly[Lys(DL-Alam)] based polymeric polypeptides are efficient against Leishmania donovani parasite infection, but the mechanism of the effect is not known yet. We prepared therefore the 5(6)-carboxyfluorescein (Cf) labelled oligopeptide [Cf-K(AaAa)]† and the correspoding MTX-conjugates [Cf-K(MTX-AaAa)] as model compounds for structure-activity experiments. The conjugate aimed to be synthesized with solid-phase methodology on MBHA resin with Boc strategy, using Fmoc-Lys(Boc)-OH. However, various side-reactions were identified. Here we report three problems observed during the synthesis as well as solutions developed by us: a) Unexpected cyclopeptide-formation with the lactone-carboxylic group of the Cf was detected, when Cf was attached to the α-amino group of the Lys residue on solid phase. This was avoided by changing the order of Cf incorporation with using Fmoc/Dde strategy. Alternatively, we have built the peptide with Fmoc strategy on solid phase first and performed the labelling with Cf-OSu subsequently in solution. b) During HF cleavage of the protected conjugates, MTX was demonstrated to form adducts with anisole and p-cresol scavengers, and the TMSOTf cleavage methodology was also found to be inadequate due to the large number of side products formed. We report here that using Fmoc/Dde strategy is an appropriate method to circumvent the cleavage with HF or TMSOTf. c) During the coupling of MTX with oligopeptide, structural and stereo isomers are formed. We have described here the suitable conditions of HPLC separation of these products

The effect of the branched chain polypeptide carrier on biodistribution of covalently attached B-cell epitope peptide (APDTRPAPG) derived from mucin 1 glycoprotein

In order to establish structure–function relationship for the design of a new group of oligopeptide antigen–macromolecule conjugate, multiple copies of mucin-1 B-cell epitope peptide, APDTRPAPG were conjugated with branched chain polymeric polypeptides possessing poly[L-Lys] backbone. By the synthesis, radiolabelling (125I) and in vivo treatment of BALB/c mice with epitope conjugates containing XiK/XAK type carrier, where X = Glu (EiK or EAK) or Leu (LAK), the influence of the polypeptide structure on the blood clearance profile and on tissue distribution profile concerning the epitope delivery to relevant organs (e.g. immunocompetent or involved in excretion) were investigated. We observed significant differences in the blood clearance profiles for the conjugates, the respective polypeptide carriers and free epitope peptide. All conjugates, regardless of their charge properties exhibited longer presence in the circulation than the free oligopeptide. Tissue distribution data also showed that the structural properties (e.g. amino acid composition, charge) of the carrier polypeptide have marked influence on the tissue accumulation of the epitope peptide conjugates. In contrast to conjugates with linear (K) or branched chain (LAK) polycationic polymers exhibiting rapid blood clearance and high spleen/liver uptake, amphoteric epitope peptide conjugates with different branches, but similar charge properties (EiK or EAK) had extended blood survival and generally lower tissue accumulation. The results on this systematic investigation suggest that further studies on the immune response induced by these epitope conjugates would be needed to provide correlation between biodistribution properties (presence in the blood, level of tissue accumulation) and the capacity of these conjugates to elicit antibody production

p25, egy új agyi fehérje szerkezete és funkciója = Structure and function of a new brain-specific protein, p25

Kutatócsoportom az elmúlt 4 évben egy új agy-specifikus fehérje szerkezeti és funkcionális jellemzésében ért el jelentős eredményeket, melyeket egyrészt magas impakt faktorú nemzetközi folyóiratokban publikáltunk, másrészt hazai és nemzetközi konferenciákon mutattunk be. Kutatásaink tárgya a TPPP/p25 fehérje volt, melyet a fehérje funkciója (Tubulin Polymerization Promoting Protein) és molekulatömege alapján neveztünk el. Megállapítottuk, hogy a fehérje szerkezet nélküli, és a különösen rendezetlen N-terminális szakasznak meghatározó szerepe van a fehérje funkciójában, mely elsődlegesen a mikrotubuláris rendszer dinamikájának szabályozásában nyilvánul meg. A fehérje N-terminális szakaszán ERK2 protein kináz általi foszforiláció szintén befolyásolja a fehérje aktivitását. Két homológját, a p20-at és p18-at, azonosítottuk, klónoztuk és jellemeztük különböző szerveződési szinteken. Megállapítottuk, hogy a TPPP fehérjék tubulinnal való kölcsönhatásához alapvető jelentőségű az egész fehérjére jellemző "unfolded" karakter. Bizonyítottuk, hogy a TPPP/p25 felhalmozódik agyszöveti zárványtestekben, melyek a szinukleionopátiák esetén alakulnak ki. Olyan antitesteket állítottunk elő, melyek specifikusan jelzik a normál agyszöveti sejteket illetve a zárványtesteket, ami rendkívüli jelentőséggel bír humán patológiai kutatásokban, valamint biomarkerként szolgálhat idegrendszeri kórképek azonosítására. | My research group was succeeded in the characterization of a new brain-specific protein in the last 4 years; the results were reported, in international journals with high impact factor and in national and international conferences. The objective of our work was the TPPP/p25 protein, which we denoted to Tubulin Polymerization Promoting Protein on the basis of its function. We showed that this protein did not have well-defined 3D structure; the unfolded N-terminal segment displayed dominant role in the protein function which was primarily manifested itself in the regulation of the dynamics of microtubule system. The phosphorylation of the N-terminal part by ERK2 protein kinase plays important role in this regulation of TPPP/p25 function. We identified and cloned two homologues of TPPP/p25. We revealed that the flexibility of the TPPP/p25 was required to express its effect on the dynamics of the microtubule system. We provided evidence that TPPP/p25 was enriched in the inclusions of brain tissues characteristic for synucleinopathies. We raised specific antibodies in rats which immunostained normal brain tissues or inclusions that could be apply in human pathological research, and they could serve as biomarkers for identification of neurodegenerative diseases

Tailoring Uptake Efficacy of HSV-1 gD Tailoring Uptake Efficacy of Hsv-1 GD Derived Carrier Peptides

Regions of the Herpes simplex virus-1 (HSV-1) glycoprotein D (gD) were chosen to design carrier peptides based on the known tertiary structure of the virus entry receptor complexes. These complexes consist of the following: HSV-1 gD–nectin-1 and HSV-1 gD–herpesvirus entry mediator (HVEM). Three sets of peptides were synthesised with sequences covering the (i) N-terminal HVEM- and nectin-1 binding region -5–42, (ii) the 181–216 medium region containing nectin-1 binding sequences and (iii) the C-terminal nectin-1 binding region 214–255. The carrier candidates were prepared with acetylated and 5(6)-carboxyfluorescein labelled N-termini. The peptides were chemically characterised and their conformational features in solution were also determined. In vitro internalisation profile and intracellular localisation were evaluated on SH-SY5Y neuroblastoma cells. Peptide originated from the C-terminal region 224–247 of the HSV-1 gD showed remarkable internalisation compared to the other peptides with low to moderate entry. Electronic circular dichroism secondary structure studies of the peptides revealed that the most effectively internalised peptides exhibit high helical propensity at increasing TFE concentrations. We proved that oligopeptides derived from the nectin-1 binding region are promising candidates—with possibility of Lys237Arg and/or Trp241Phe substitutions—for side-reaction free conjugation of bioactive compounds—drugs or gene therapy agents—as cargos

The mapping of linear B-cell epitope regions in desmoglein 1 and 3 proteins : Recognition of immobilized peptides by pemphigus patients’ serum autoantibodies

Desmosomal transmembrane glycoproteins desmoglein 1and desmoglein 3 are targets of life-threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus(PF). In these diseases pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell-cell adhesion, and clinically, to the presence of blisters and erosions. Identification, characterization and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding theimmunopathology of the disease and also in the design of novel diagnostics. Here we describe the localization of the B-cell epitope regions of Dsg1 and Dsg3 proteins extracellular parts recognized by IgG-type serum autoantibodies of patients with PV andPF. In our study overlapping pentadecapeptides were synthesized on hydroxypropylmethacrylate pins based on the results of in silicopredictions. To detect the interaction between theserum autoantibodies and the immobilized synthetic peptides, modified ELISA (Enzyme Linked Immunosorbent Assay) was performed with pin-attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86-110, aa196-220, aa226-250, aa326-340, and aa486-520) within the Dsg1 protein sequence and four possible epitope regions (aa64-78, aa330-344, aa375-399, aa446-460) within the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin-bound overlapping peptides in modified ELISAs

Cellular Uptake Mechanism of Cationic Branched Polypeptides with Poly[L‑Lys] Backbone

Cationic macromolecular carriers can be effective carriers for small molecular compounds, drugs, epitopes, or nucleic acids. Polylysine-based polymeric branched polypeptides have been systematically studied on the level of cells and organisms as well. In the present study, we report our findings on the cellular uptake characteristics of nine structurally related polylysine-based polypeptides with cationic side chains composed of (i) single amino acid (poly[Lys(Xi)], XiK) or (ii) oligo[DL-alanine] (poly[Lys(DL-Alam)], AK) or (iii) oligo[DL-alanine] with an additional amino acid (X) at the terminal position (poly[Lys(Xi-DL-Alam)] (XAK)) or (iv) at the position next to the polylysine backbone (poly[Lys(DL-Alam-Xi)] (AXK)). In vitro cytotoxicity and cellular uptake were characterized on HT-29 human colon carcinoma and HepG2 human hepatocarcinoma cell lines. Data indicate that the polycationic polypeptides studied are essentially nontoxic in the concentration range studied, and their uptake is very much dependent on the side chain structure (length, identity of amino acid X, and distance between the terminal positive charges) and also on the cell lines. Our findings in uptake inhibition studies suggest that predominantly macropinocytosis and caveole/lipid raft mediated endocytosis are involved. The efficacy of their internalization is markedly influenced by the hydrophobicity and charge properties of the amino acid X. Interestingly, the uptake properties of the these polypeptides show certain similarities to the entry pathways of several cell penetrating peptides

Probing of primed and unprimed sites of calpains: Design, synthesis and evaluation of epoxysuccinyl-peptide derivatives as selective inhibitors

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 NH 2-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. © 2014 Elsevier Masson SAS. All rights reserved

NaBH4 – a novel method for the deprotection of Nω-nitro-arginine

The selective deprotection of Nω-nitro-arginine derivatives represents a major preparative challenge. This problem can be circumvented by the use of catalytic hydrogenation, but often high pressure, elevated temperature, and/or long reaction times are needed. In certain cases hydrogenation is not suitable, for example, small-scale reactions, parallel synthesis, or due to selectivity issues. Herein, we demonstrate for the first time, the use of NaBH4 in the presence of a metal ion catalyst for the removal of the Nω-nitro moiety under simple, ‘open-vessel’ conditions. This process using NaBH4 does not remove the benzyloxy-carbonyl-protecting group; thus the method is orthogonal for this protecting scheme