Cell-penetrating conjugates of pentaglutamylated methotrexate as potential anticancer drugs against resistant tumor cells.

The emerging resistance of tumor cells against methotrexate (MTX) is one of the major limitations of the MTX treatment of tumorous diseases. The disturbance in the polyglutamation which is a main step in the mechanism of methotrexate action is often the reason of the resistance. Delivery of polyglutamylated MTX into cells may evade the mechanisms that are responsible for drug resistance.

Comparative in vitro biological evaluation of daunorubicin containing GnRH-I and GnRH-II conjugates developed for tumor targeting.

Hormone based drug targeting is a promising tool for selective tumor therapy. In this study, synthesis and systematic comparative biological evaluation of novel drug containing analogs of gonadotropin-releasing hormone GnRH-I and GnRH-II is reported demonstrating their suitability for tumor targeting. The cytotoxic conjugates were prepared by the attachment of the chemotherapeutical agent daunorubicin (Dau) to GnRH analogs directly or through an enzyme-labile spacer with oxime linkage.

Modification of daunorubicin-GnRH-III bioconjugates with oligoethylene glycol derivatives to improve solubility and bioavailability for targeted cancer chemotherapy.

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.

Tritium labelling of a cholesterol amphiphile designed for cell membrane anchoring of proteins.

Cell membrane association of proteins can be achieved by the addition of lipid moieties to the polypeptide chain, and such lipid-modified proteins have important biological functions. A class of cell surface proteins contains a complex glycosylphosphatidylinositol (GPI) glycolipid at the C-terminus, and they are accumulated in cholesterol-rich membrane microdomains, that is, lipid rafts. Semisynthetic lipoproteins prepared from recombinant proteins and designed lipids are valuable probes and model systems of the membrane-associated proteins.

Cell-free expression of G-protein-coupled receptors.

Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives.

Co-translational stabilization of insoluble proteins in cell-free expression systems.

Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment.

Cell-free expression of G-protein coupled receptors: new pipelines for challenging targets.

Based on their eminent importance for medical applications, G-protein coupled receptors are currently amongst the most frequently membrane protein targets analyzed by cell-free expression. The cell-free expression approach removes most bottlenecks known from conventional cell-based protein production pipelines and ensures fast access to a selected receptor target. In addition, receptors can be synthesized in presence of a large variety of artificial solubilization environments comprising detergents, lipids, nanodiscs and other amphiphilic compounds.

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da  = Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice.

Preparation of semisynthetic lipoproteins with fluorescent cholesterol anchor and their introduction to the cell membrane with minimal disruption of the membrane.

The exogenous introduction of fluorescent lipoproteins into cell membranes is a method for visualizing the cellular traffic of membrane associated proteins, and also for altering the cell surface in a controlled manner. In order to achieve the cell membrane anchoring of proteins and their subsequent fluorescence based detection, a cholesterol derivative was designed. The headgroup of the novel cholesterol anchor contains a fluorescent reporter and a thiol reactive maleimide for protein conjugation.

Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments.

The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs.

Dehydroepiandrosterone post-transcriptionally modifies CYP1A2 induction involving androgen receptor.

The pharmacological dosage of dehydroepiandrosterone (DHEA) protects against chemically induced carcinogenesis. The chemoprotective activity of DHEA is attributed to its inhibitory potential for the expression of CYP1A enzymes, which are highly responsible for metabolic activation of several mutagenic and carcinogenic chemicals. The present work investigated whether the chemoprevention by DHEA was due to diminished transcriptional activation of CYP1A genes or to the post-transcriptional modulation of CYP1A expression.

Synthesis, enzymatic stability and in vitro cytostatic effect of Daunorubicin-GnRH-III derivative dimers.

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(DauAoa-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.

Enhanced cellular uptake and in vitro antitumor activity of short-chain fatty acid acylated daunorubicin-GnRH-III bioconjugates.

Here we report on the synthesis and biochemical characterization (enzymatic stability, cellular uptake, in vitro antitumor activity, membrane interaction and GnRH-receptor binding affinity) of novel short-chain fatty acid (SCFA) acylated daunorubicin-GnRH-III bioconjugates, which may serve as drug delivery systems for targeted cancer chemotherapy. Ser in position 4 of GnRH-III was replaced by Lys, followed by the acylation of its ε-amino group with various fatty acids. SCFAs are potentially chemoprotective agents by suppressing the growth of cancer cells and therefore may enhance the antitumor activity of the bioconjugates.

Synthesis and cytotoxic activity of novel 5-substituted-1-(β-L-arabinofuranosyl) pyrimidine nucleosides.

A series of new 5-halogeno-1-(ß-L-arabinofuranosyl)uracils and their cytosine analogues were synthesized by halogenation of ara-L-uridine and ara-L-cytidine, respectively. The 5-(2-thienyl) and 5-halogenothienyl derivatives of both series were also prepared in excellent yields by Stille coupling followed by halogenation. All of these syntheses were based on benzoyl-protected derivatives.

GnRH-III based multifunctional drug delivery systems containing daunorubicin and methotrexate.

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.

Acylated mono-, bis- and tris- cinchona-based amines containing ferrocene or organic residues: synthesis, structure and in vitro antitumor activity on selected human cancer cell lines.

A series of novel functionalized mono-, bis- and tris-(S)-{[(2S,4R,8R)-8-ethyl-quinuclidin-2-yl](6-methoxyquinolin-4-yl)}methanamines including ferrocene-containing derivatives was obtained by the reaction of the precursor amine with a variety of acylation agents. Their in vitro antitumor activity was investigated against human leukemia (HL-60), human neuroblastoma (SH-SY5Y), human hepatoma (HepG2) and human breast cancer (MCF-7) cells by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay and the 50% inhibitory concentration (IC(50)) values were determined. Our data indicate that the precursor amine has no antitumor activity in vitro, but the bis-methanamines with ureido-, thioureido and amide-type linkers display attractive in vitro cytotoxicity and cytostatic effects on HL-60, HepG2, MCF-7 and SH-SY5Y cells.

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.

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.

Anthracycline-GnRH derivative bioconjugates with different linkages: synthesis, in vitro drug release and cytostatic effect.

To increase the selectivity and consequently to minimize the side effects of chemotherapeutic agents, receptor mediated tumor targeting approaches have been developed. In the present work, various anthracycline-GnRH derivative bioconjugates were synthesized with the aim of investigating the influence of (i) different anthracycline anticancer drugs, (ii) different linkages between the targeting moiety and the anticancer drug, and (iii) different targeting moieties (e.g.

Enhanced enzymatic stability and antitumor activity of daunorubicin-GnRH-III bioconjugates modified in position 4.

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), (4)Ser was replaced by N-Me-Ser or Lys(Ac). A compound in which (4)Lys was not acetylated was also prepared, with the aim of investigating the influence of the free ε-amino group on the biochemical properties.

Design, synthesis, in vitro stability and cytostatic effect of multifunctional anticancer drug-bioconjugates containing GnRH-III as a targeting moiety.

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.

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.

In vitro degradation and antitumor activity of oxime bond-linked daunorubicin-GnRH-III bioconjugates and DNA-binding properties of daunorubicin-amino acid metabolites.

Bioconjugates with receptor-mediated tumor-targeting functions and carrying cytotoxic agents should enable the specific delivery of chemotherapeutics to malignant tissues, thus increasing their local efficacy while limiting the peripheral toxicity. In the present study, gonadotropin-releasing hormone III (GnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2)) was employed as a targeting moiety to which daunorubicin was attached via oxime bond, either directly or by insertion of a GFLG or YRRL tetrapeptide spacer. The in vitro antitumor activity of the bioconjugates was determined on MCF-7 human breast and HT-29 human colon cancer cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.

Efficient synthesis of an (aminooxy) acetylated-somatostatin derivative using (aminooxy)acetic acid as a 'carbonyl capture' reagent.

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated-somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a 'carbonyl capture' reagent in the final cleavage step.