Zoltán Bánóczi

Novel Cell-Penetrating Calpain Substrate

The calpain enzymes play important roles in numerous processes in the cell. In vivo analysis of calpain activity might be useful for clarification of their role in different diseases. Our early results suggested that a peptide substrate, Dabcyl-TPLKSPPPSPR- EDANS, based on the calpain cleavage sequences is suitable for developing a new cell-penetrating calpain substrate. This conjugate with the Dabcyl and EDANS fluorophores as a FRET pair is specific for calpain even in cell lysate, but unfortunately has poor cell uptake. Therefore, we have modified this sequence by C-terminal elongation with heptaarginine unit possessing cell-penetrating activity. In order to preserve the necessary distance between the two FRET partners, we inserted a Glu residue between the substrate and heptaarginine parts of the peptide. Thus, the cell-penetrating substrate Dabcyl-TPLKSPPPSPRE( EDANS)R 7 was synthesized. This peptide not only retained the substrate property, but was a better substrate of Calpain B enzyme. The cell uptake of the substrate conjugate was studied by fluorescence microscopy and flow cytometry. The results showed that the conjugate enters COS-7 cells more efficiently than the peptide substrate without heptaarginine. The uptake occurs already at low concentration and the compound is distributed homogeneously inside cells. These observations might indicate that this new cell-penetrating substrate could be useful for determining calpain activity in cell lysate or in intact cells of various origins.

A kinked antimicrobial peptide from Bombina maxima. I. Three-dimensional structure determined by NMR in membrane-mimicking environments

Maximin-4 is a 27-residue cationic antimicrobial peptide exhibiting selectivity for bacterial cells. As part of the innate defense system in the Chinese red-belly toad, its mode of action is thought to be ion channel or pore formation and dissipation of the electrochemical gradient across the pathogenic cell membrane. Here we present the high-resolution structure of maximin-4 in two different membrane mimetics, sodium dodecyl sulfate micelles and 50% methanol, as determined by 1H solution NMR spectroscopy. In both environments, the peptide chain adopts a helix–break–helix conformation following a highly disordered N-terminal segment. Despite the similarities in the overall topology of the two structures, major differences are observed in terms of the interactions stabilizing the kink region and the arrangement of the four lysine residues. This has a marked influence on the shape and charge distribution of the molecule and may have implications for the bacterial selectivity of the peptide. The solution NMR results are complemented by CD spectroscopy and solid-state NMR experiments in lipid bilayers, both confirming the predominantly helical conformation of the peptide. As a first step in elucidating the membrane interactions of maximin-4, our study contributes to a better understanding of the mode of action of antimicrobial peptides and the factors governing their selectivity.

Synthesis and in vitro antitumor effect of vinblastine derivative-oligoarginine conjugates.

Vinblastine is a widely used anticancer drug with undesired side effects. Its conjugation with carrier molecules could be an efficient strategy to reduce these side effects. Besides this, the conjugate could exhibit increased efficiency against resistant cells, e.g., due to the altered internalization pathway. Oligoarginines, as cell-penetrating peptides, can transport covalently attached compounds into different kinds of cells and enhance the efficiency of those compounds. We report here the coupling of vinblastine through its carboxyl group at position 16 with the N-terminal amino function of L-Trp methyl ester. After hydrolysis of the ester group, 17-desacetylvinblastineTrp was conjugated to the N-terminal amino group of oligoarginine via the C-terminal carboxyl group of the Trp moiety in solution. The antitumor effect of conjugates was studied on sensitive and resistant human leukemia (HL-60) cells in vitro. Our data suggest that all conjugates investigated possess an antiproliferative effect against the studied cells. However, the effect was dependent on the number of Arg residues in the conjugates: Arg₈ > Arg₆ ≫ Arg₄. The conjugate with Arg₈ exhibited similar efficicacy as compared with free 17-desacetylvinblastineTrp. The in vitro studies also showed that the tubulin binding ability of vinblastine was essentially preserved even in the octaarginine conjugate. We also observed that two isomers were formed during conjugation. These isomers showed different levels of activity against tubulin polymerization in vitro and in vivo. The 17-desacetylvinblastineTrp-Arg₈-1 isomer conjugate possessed high selectivity against the mitotic spindles. HRMS and NMR data suggest that 17-desacetylvinblastineTrp-Arg₈-1 and 17-desacetylvinblastineTrp-Arg₈-2 are epimers at the tryptophan α carbon atom.

A New Daunomycin–Peptide Conjugate: Synthesis, Characterization and the Effect on the Protein Expression Profile of HL-60 Cells in Vitro

Daunomycin (Dau) is a DNA-binding antineoplastic agent in the treatment of various types of cancer, such as osteosarcomas and acute myeloid leukemia. One approach to improve its selectivity and to decrease the side effects is the conjugation of Dau with oligopeptide carriers, which might alter the drug uptake and intracellular fate. Here, we report on the synthesis, characterization, and in vitro biological properties of a novel conjugate in which Dau is attached, via an oxime bond, to one of the cancer specific small peptides (LTVSPWY) selected from a random phage peptide library. The in vitro cytostatic effect and cellular uptake of Dau═Aoa-LTVSPWY-NH(2) conjugate were studied on various human cancer cell lines expressing different levels of ErbB2 receptor which could be targeted by the peptide. We found that the new daunomycin-peptide conjugate is highly cytostatic and could be taken up efficiently by the human cancer cells studied. However, the conjugate was less effective than the free drug itself. RP-HPLC data indicate that the conjugate is stable at least for 24 h in the pH 2.5-7.0 range of buffers, as well as in cell culture medium. The conjugate in the presence of rat liver lysosomal homogenate, as indicated by LC-MS analysis, could be degraded. The smallest, Dau-containing metabolite (Dau═Aoa-Leu-OH) identified and prepared expresses DNA-binding ability. In order to get insight on the potential mechanism of action, we compared the protein expression profile of HL-60 human leukemia cells after treatment with the free and peptide conjugated daunomycin. Proteomic analysis suggests that the expression of several proteins has been altered. This includes three proteins, whose expression was lower (tubulin β chain) or markedly higher (proliferating cell nuclear antigen and protein kinase C inhibitor protein 1) after administration of cells with Dau-conjugate vs free drug.

Daunomycin-polypeptide conjugates with antitumor activity

We have developed a group of water-soluble drug conjugates in which daunomycin (Dau) is coupled to cationic, amphoteric or anionic branched polypeptides and a new conjugate containing a cationic polypeptide carrier modified with a cell penetrating octaarginine. We investigated in vitro physiological activity of these conjugates in several aspects: in vitro cytotoxicity and cytostatic effect, adhesion and cellular uptake were examined on murine (J774 and L1210) and human (MonoMac6 and HL-60) leukemia cell lines and on murine bone marrow derived macrophages. We found that these processes are dependent on the properties of the carrier, on experimental conditions like concentration and incubation time. We found that attachment of polypeptide and cell penetrating peptide to the bioactive agent, depending on the cell line, could significantly improve the antitumor activity of the drug.

New pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-60) cells

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non‐small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem‐peptide conjugates in which cell‐penetrating octaarginine or/and lung‐targeting H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH2 peptide is attached to the drug by thioether bond. The conjugates characterized by RP‐HPLC and MS exhibited cytostatic effect in vitro on non‐small cell lung carcinoma as well as on human leukemia cell lines. The IC50 values of the conjugates were similar, but the conjugates with H‐Ile‐Glu‐Leu‐Leu‐Gln‐Ala‐Arg‐NH2 sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell‐penetrating or cell‐targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates. Copyright

Synthesis and in vitro antitumor effect of new vindoline derivatives coupled with amino acid esters

10-Bromovindoline and its 14,15-dihydro- and 14,15-cyclopropano derivatives were coupled in the position 16 with (L)- and (D)-tryptophan methyl esters. The tryptophan derivatives of vindoline were synthesized starting from the 16-carboxylic acid hydrazides viathe corresponding azides which were allowed to react with the amino acidesters. The new compounds showed antitumor activity against human leukemia (HL-60) cells in vitro.

Synthetic calpain activator boosts neuronal excitability without extra Ca2

Earlier we have shown that an equimolar mixture of calpastatin subdomains A and C (19 amino acids each) strongly activates m-calpain in vitro. In the present work we developed a membrane-permeable activator system, by conjugating an oligo-arginine tail to both peptides. We tested calpain activation as well as synaptic excitability on rat brain slices ex vivo. In hippocampal slices both basic excitability and long-term synaptic efficacy were significantly increased upon treatment with the activator. We propose that the activator peptide conjugates can be used with any mammalian cell, to specifically challenge the calpain system apparently without raising cytoplasmic Ca2+. Such an effector may be a useful tool in dissecting intracellular mechanisms involving the calpain system.

New m-calpain substrate-based azapeptide inhibitors

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. Copyright

Folding transitions in calpain activator peptides studied by solution NMR spectroscopy

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 Ca2+ 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% CD3OH. 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 Ca2+ 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. Copyright