2015, Hegedüs, Rózsa, Pauschert, Aline, Orbán, Erika, Szabó, Ildikó, Andreu, David, Marquardt, Andreas, Mező, Gábor, Manea, Marilena

Daunorubicin-GnRH-III bioconjugates have recently been developed as drug delivery systems with potential applications in targeted cancer chemotherapy. In order to improve their biochemical properties, several strategies have been pursued: (1) incorporation of an enzymatic cleavable spacer between the anticancer drug and the peptide-based targeting moiety, (2) peptide modification by short chain fatty acids or (3) attachment of two anticancer drugs to the same GnRH-III derivative. Although these modifications led to more potent bioconjugates, a decrease in their solubility was observed. Here we report on the design, synthesis and biochemical characterization of daunorubicin-GnRH-III bioconjugates with increased solubility, which could be achieved by incorporating oligoethylene glycol-based spacers in their structure. First, we have evaluated the effect of an oligoethylene glycol-based spacer on the solubility, enzymatic stability/degradation, cellular uptake and in vitro cytostatic effect of a bioconjugate containing only one daunorubicin attached through a GFLG tetrapeptide spacer to the GnRH-III targeting moiety. Thereafter, more complex compounds containing two copies of daunorubicin, GFLG spacers as well as Lys(nBu) in position 4 of GnRH-III were synthesized and biochemically characterized. Our results indicated that all synthesized oligoethylene glycol-containing bioconjugates had higher solubility in cell culture medium than the unmodified analogs. They were degraded in the presence of rat liver lysosomal homogenate leading to the formation of small drug containing metabolites. In the case of bioconjugates containing two copies of daunorubicin, the incorporation of oligoethylene glycol-based spacers led to increased in vitro cytostatic effect on MCF-7 human breast cancer cells.

2012, Leurs, Ulrike, Mező, Gábor, Orbán, Erika, Öhlschläger, Peter, Marquardt, Andreas, Manea, Marilena

Bioconjugates containing the GnRH-III hormone decapeptide as a targeting moiety are able to deliver chemotherapeutic agents specifically to cancer cells expressing GnRH receptors, thereby increasing their local efficacy while limiting the peripheral toxicity. However, the number of GnRH receptors on cancer cells is limited and they desensitize under continuous hormone treatment. A possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of the bioconjugates would be the attachment of more than one chemotherapeutic agent to one GnRH-III molecule.

Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing GnRH-III as a targeting moiety and daunorubicin as a chemotherapeutic agent. Two different drug design approaches were pursued. The first one was based on the bifunctional [4Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH2) containing two lysine residues in positions 4 and 8, whose ε-amino groups were used for the coupling of daunorubicin. In the second drug design, the native GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2) was used as a scaffold; an additional lysine residue was coupled to the ε-amino group of 8Lys in order to generate two free amino groups available for conjugation of daunorubicin. The in vitro stability/degradation of all synthesized compounds was investigated in human serum, as well as in the presence of rat liver lysosomal homogenate. Their cellular uptake was determined on human breast cancer cells and the cytostatic effect was evaluated on human breast, colon and prostate cancer cell lines.

Compared to a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.

2009, Jung, Ji Eun, Wollscheid, Hans-Peter, Marquardt, Andreas, Manea, Marilena, Scheffner, Martin, Przybylski, Michael

The modification of ubiquitin to defined oligo-ubiquitinated conjugates has received considerable interest due to the finding that isomeric oligo-ubiquitin conjugates exhibit distinct differences in their biochemical functions, depending on the specific lysine-ε-amino linkage used for conjugate formation. Here, we report the design and development of a thioether linkage-based approach for the synthesis of oligo-ubiquitin conjugates with lysine-specific branching by thioether ligation of a linear ubiquitin peptide containing a C-terminal cysteine residue as the "donor" component, with a corresponding lysine-ε-amino-branched haloacyl-activated ubiquitin "acceptor" peptide. This approach was successfully used for the synthesis of a lysine-63-linked diubiquitin conjugate by ligation of the modified ubiquitin(1-52)-Cys- donor peptide to the N-terminal Arg-54 residue of the branched Lys-63-linked acceptor peptide, ubiquitin(54-76)2. Advantages of the present approach are as follows: (i) the conjugation reaction is performed in solution using suitable preformed donor ubiquitin peptides with a C-terminal Cys residue, and (ii) different corresponding N-chloroacetylated ubiquitin acceptor peptides containing the branched Lys residue are employed, providing broad applicability to the preparation of isomeric oligo-ubiquitin conjugates. The Lys-63-diubiquitin conjugate 7 described here was purified by semipreparative HPLC, and its structure and homogeneity ascertained by HPLC and high-resolution MALDI and electrospray-mass spectrometry. CD spectra and molecular modeling indicate a conformationally stable structure of the conjugate with spatial separation of the ubiquitin parts of the Lys-63 linkage. Moreover, the activity of the thioether-linked diubiquitin conjugate was ascertained by in vitro autoubiquitination assay. These results indicate the feasibility of this approach for the preparation of functional oligo-ubiquitin conjugates.

2013-04-01, Schreier, Verena Natalie, Mező, Gábor, Orbán, Erika, Dürr, Claudia, Marquardt, Andreas, Manea, Marilena

Bioconjugates containing chemotherapeutic agents attached to peptide hormones, such as gonadotropin-releasing hormone (GnRH), are developed as drug delivery systems for targeted cancer chemotherapy. We report here the synthesis and biochemical characterization of disulfide bond-linked dimeric bioconjugates in which daunorubicin was coupled via an oxime linkage to aminooxyacetylated GnRH-III ([Glp-His-Trp-Ser-His-Asp-Trp-Lys(Dau=Aoa-Cys)-Pro-Gly-NH2]2; where Glp is pyroglutamic acid and Aoa is aminooxyacetyl) and its derivatives modified in position four by N-Me-Ser and Lys(Ac). The in vitro stability/degradation of the bioconjugates was determined in human serum, as well as in the presence of rat liver lysosomal homogenate and digestive enzymes. All compounds were stable at least for 24h in human serum and in the presence of pepsin and trypsin, while they were degraded by lysosomal enzymes. The daunorubicin-GnRH-III derivative dimers were partly digested by α-chymotrypsin; however, they had increased stability compared to the corresponding monomers, making them potential candidates for oral administration. The in vitro cytostatic effect of the compounds was determined on MCF-7 human breast cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. All daunorubicin-GnRH-III derivative dimers exerted slightly increased in vitro cytostatic effect (IC50 values in low μM range) than the corresponding monomeric bioconjugates.

2011-10-19, Orbán, Erika, Manea, Marilena, Marquardt, Andreas, Bánóczi, Zoltán, Csı́k, Gabriella, Fellinger, Erzsébet, Bősze, Szilvia, Hudecz, Ferenc

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-NH2 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.

2007, Marquardt, Andreas, Bernevic, Bogdan, Przybylski, Michael

A cyclic disulfide heptadecapeptide (TIP17ox; 2) derived from the lectin-like 17-amino acid domain of human tumor necrosis factor-α [TNF-α (100-116)] was synthesised and demonstrated to bind specifically to N,N-diacetylchitobiose, a disaccharide present in many glycan structures of glycoproteins. Although the TIP domain forms a loop structure in the native TNF-α protein, we show in this study by high-resolution ESI-FTICR mass spectrometry that a homologous linear heptadecapeptide (TIP17rd; 1) binds with comparable affinity to chitobiose, suggesting that cyclisation is not essential for carbohydrate binding. ESI-FTICR-MS was used as an efficient tool for the direct molecular characterisation of TIP peptide-carbohydrate complexes. The specific binding of the TNF-TIP domain to chitobiose and other carbohydrate motifs in glycoproteins may explain the high proteolytic stability of these peptides in biological fluids. A considerably higher proteolytic stability in human plasma was found by mass spectrometric analysis for the cyclic TIP peptide 2, compared to the linear peptide 1. Furthermore, affinity-proteomics studies using immobilised cyclic TIP peptide 2 provided the identification of specific interacting glycoproteins in plasma.

2012-06, Leurs, Ulrike, Lajkó, Eszter, Mező, Gábor, Orbán, Erika, Öhlschläger, Peter, Marquardt, Andreas, Kőhidai, László, Manea, Marilena

Here we report on the design, synthesis and biochemical characterization of multifunctional bioconjugates containing two chemotherapeutic agents, daunorubicin and methotrexate, coupled to the GnRH-III decapeptide, which served as a targeting moiety. This represents a possible approach to increase the receptor mediated tumor targeting and consequently the cytostatic effect of anticancer drug-peptide bioconjugates. The multifunctional bioconjugates were prepared according to two drug design approaches recently developed by our group. Both bifunctional GnRH-III derivatives, [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) and [(8)Lys(Lys)]-GnRH-III (Glp-His-Trp-Ser-His-Asp-Trp-Lys(Lys)-Pro-Gly-NH(2)), contain two free amino groups suitable for the attachment of two anticancer drugs, such as methotrexate and daunorubicin. The drugs were chosen with respect to their different mechanisms of action, with the goal of increasing the antitumor effect of the bioconjugates. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their in vitro stability/degradation in human serum and in the presence of rat liver lysosomal homogenate was investigated by liquid chromatography in combination with mass spectrometry. The influence of the multifunctional bioconjugates on the cell adhesion and cell proliferation was studied on Mono Mac 6 human leukemic monocytes. It was found that (1) all synthesized bioconjugates had in vitro cytostatic effect; (2) they were stable in human serum for at least 24 h; (3) they were hydrolyzed in the presence of lysosomal homogenate and (4) they exerted a moderate cell-cell adhesion inducing effect. These results demonstrate that multifunctional bioconjugates containing two different anticancer drugs attached to the same GnRH-III targeting moiety could be successfully prepared and resulted in higher in vitro cytostatic effect than the monofunctional bioconjugates containing either methotrexate or daunorubicin, in particular on HT-29 human colon cancer cells.

2011, Manea, Marilena, Leurs, Ulrike, Orbán, Erika, Baranyai, Zsuzsa, Öhlschläger, Peter, Marquardt, Andreas, Schulcz, Ákos, Tejeda, Miguel, Kapuvári, Bence, Tóvári, József, Mező, Gábor

Here, we report on the synthesis, enzymatic stability, and antitumor activity of novel bioconjugates containing the chemotherapeutic agent daunorubicin attached through an oxime bond to various gonadotropin-releasing hormone-III (GnRH-III) derivatives. In order to increase the enzymatic stability of the bioconjugates (in particular against chymotrypsin), 4Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which 4Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties. The in vitro cytostatic effect of the bioconjugates was determined on MCF-7 human breast, HT-29 human colon, and LNCaP human prostate cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Their stability/degradation (1) in human serum, (2) in the presence of rat liver lysosomal homogenate, and (3) in the presence of digestive enzymes (trypsin, chymotrypsin, and pepsin) was analyzed by liquid chromatography in combination with mass spectrometry. The results showed that (1) all synthesized bioconjugates had in vitro cytostatic effect, (2) they were stable in human serum at least for 24 h, and (3) they were hydrolyzed in the presence of lysosomal homogenate. All compounds were stable in the presence of (1) pepsin and (2) trypsin (except for the 4Lys containing bioconjugate). In the presence of chymotrypsin, all bioconjugates were digested; the degradation rate strongly depending on their structure. The bioconjugates in which 4Ser was replaced by N-Me-Ser or Lys(Ac) had the highest enzymatic stability, making them potential candidates for oral administration. In vivo tumor growth inhibitory effect of two selected bioconjugates was evaluated on orthotopically developed C26 murine colon carcinoma bearing mice. The results indicated that the compound containing Lys(Ac) in position 4 had significantly higher antitumor activity than the parent bioconjugate.

2006, Marquardt, Andreas