RESUMEN
The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Apoptosis/efectos de los fármacos , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/farmacología , Animales , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/genética , Biología Computacional/métodos , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacología , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Humanos , TranscriptomaRESUMEN
Background: Peptide hormone-based targeted tumor therapy is an approved strategy to selectively block the tumor growth and spreading. The gonadotropin-releasing hormone receptors (GnRH-R) overexpressed on different tumors (e.g., melanoma) could be utilized for drug-targeting by application of a GnRH analog as a carrier to deliver a covalently linked chemotherapeutic drug directly to the tumor cells. In this study our aim was (i) to analyze the effects of GnRH-drug conjugates on melanoma cell proliferation, adhesion and migration, (ii) to study the mechanisms of tumor cell responses, and (iii) to compare the activities of conjugates with the free drug. Results: In the tested conjugates, daunorubicin (Dau) was coupled to 8Lys of GnRH-III (GnRH-III(Dau=Aoa)) or its derivatives modified with 4Lys acylated with short-chain fatty acids (acetyl group in [4Lys(Ac)]-GnRH-III(Dau=Aoa) and butyryl group in [4Lys(Bu)]-GnRH-III(Dau=Aoa)). The uptake of conjugates by A2058 melanoma model cells proved to be time dependent. Impedance-based proliferation measurements with xCELLigence SP system showed that all conjugates elicited irreversible tumor growth inhibitory effects mediated via a phosphoinositide 3-kinase-dependent signaling. GnRH-III(Dau=Aoa) and [4Lys(Ac)]-GnRH-III(Dau=Aoa) were shown to be blockers of the cell cycle in the G2/M phase, while [4Lys(Bu)]-GnRH-III(Dau=Aoa) rather induced apoptosis. In short-term, the melanoma cell adhesion was significantly increased by all the tested conjugates. The modification of the GnRH-III in position 4 was accompanied by an increased cellular uptake, higher cytotoxic and cell adhesion inducer activity. By studying the cell movement of A2058 cells with a holographic microscope, it was found that the migratory behavior of melanoma cells was increased by [4Lys(Ac)]-GnRH-III(Dau=Aoa), while the GnRH-III(Dau=Aoa) and [4Lys(Bu)]-GnRH-III(Dau=Aoa) decreased this activity. Conclusion: Internalization and cytotoxicity of the conjugates showed that GnRH-III peptides could guard Dau to melanoma cells and promote antitumor activity. [4Lys(Bu)]-GnRH-III(Dau=Aoa) possessing the butyryl side chain acting as a "second drug" proved to be the best candidate for targeted tumor therapy due to its cytotoxicity and immobilizing effect on tumor cell spreading. The applicability of impedimetry and holographic phase imaging for characterizing cancer cell behavior and effects of targeted chemotherapeutics with small structural differences (e.g., length of the side chain in 4Lys) was also clearly suggested.
RESUMEN
Compared to classical chemotherapy, peptide-based drug targeting is a promising therapeutic approach for cancer, which can provide increased selectivity and decreased side effects to anticancer drugs. Among various homing devices, gonadotropin-releasing hormone-III (GnRH-III) peptide represents a suitable targeting moiety, in particular in the treatment of hormone independent tumors that highly express GnRH receptors (e.g. colon carcinoma). We have previously shown that GnRH-III[(4)Lys(Ac),(8)Lys(Dau = Aoa)] bioconjugate, in which daunorubicin was attached via oxime linkage to the (8)Lys of a GnRH-III derivative, exerted significant in vivo antitumor effect on subcutaneously developed HT-29 colon tumor. In contrast, results of the study reported here indicated that this compound was not active on an orthotopically developed tumor. However, if Lys in position 4 was acylated with butyric acid instead of acetic acid, the resulting bioconjugate GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] had significant tumor growth inhibitory effect. Furthermore, it prevented tumor neovascularization, without detectable side effects. Nevertheless, the development of metastases could not be inhibited by the bioconjugate; therefore, its application in combination with a metastasis preventive agent might be necessary in order to achieve complete tumor remission. In spite of this result, the treatment with GnRH-III[(4)Lys(Bu),(8)Lys(Dau = Aoa)] bioconjugate proved to have significant benefits over the administration of free daunorubicin, which was used at the maximum tolerated dose.
Asunto(s)
Antibióticos Antineoplásicos/uso terapéutico , Ácido Butírico/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Daunorrubicina/uso terapéutico , Hormona Liberadora de Gonadotropina/uso terapéutico , Ácido Pirrolidona Carboxílico/análogos & derivados , Animales , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidad , Ácido Butírico/química , Ácido Butírico/toxicidad , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Daunorrubicina/química , Daunorrubicina/toxicidad , Femenino , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/toxicidad , Células HT29 , Corazón/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Masculino , Ratones , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patología , Ácido Pirrolidona Carboxílico/química , Ácido Pirrolidona Carboxílico/uso terapéutico , Ácido Pirrolidona Carboxílico/toxicidad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Daunorrubicina , Sistemas de Liberación de Medicamentos/métodos , Hormona Liberadora de Gonadotropina , Polietilenglicoles , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Daunorrubicina/química , Daunorrubicina/farmacología , Femenino , Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/farmacología , Humanos , Células MCF-7 , Polietilenglicoles/química , Polietilenglicoles/farmacología , Ratas , SolubilidadRESUMEN
Targeted tumor therapy is a perspective procedure to specifically destroy the cancer tissues with eliminating or at least decreasing the side effects of anticancer drugs. For this purpose the drug molecule is attached to a targeting moiety (e.g. peptide hormones) that recognizes tumor specific or overexpressed receptors on cancer cells. The in vitro cytostatic or cytotoxic assays do not give proper information whether the tumor growth inhibitory effect of the conjugate is better than the activity of the free drug. Only in vivo studies are adequate to answer this question. However, the selection of the appropriate tumor model is important to eliminate the false positive results. In our studies a gonadotropin-releasing hormone analog (GnRH-III) was applied as targeting moiety in drug conjugates. The in vivo antitumor activity of these conjugates was investigated on mice bearing subcutaneously or orthotopically szigdeveloped tumors. The subcutaneously implanted tumor model which is isolated from its surroundings may provide false results in tumor growth inhibition. In contrast, the orthotopically developed tumor is a better model representing appropriate anatomical and clinical status of cancer. Therefore, the orthotopical colon cancer developed in our laboratory is a suitable model for the study of the antitumor activity of the conjugates prepared for targeted tumor therapy.
RESUMEN
MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.
Asunto(s)
Serina Proteasas Asociadas a la Proteína de Unión a la Manosa , Péptidos , Disulfuros , Humanos , Lectinas , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/antagonistas & inhibidores , Serina Proteasas Asociadas a la Proteína de Unión a la Manosa/química , Péptidos/química , Péptidos/farmacologíaRESUMEN
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. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4-fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography.
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Ácido Aminooxiacético/química , Daunorrubicina/química , Somatostatina/química , Somatostatina/síntesis química , Acetilación , Línea Celular Tumoral , Cromatografía Líquida de Alta Presión , Humanos , Espectrometría de Masas , Estructura MolecularRESUMEN
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. We found that all synthesized bioconjugates had high cytostatic effect in vitro, were stable in cell culture medium for 6 h and degraded in the presence of rat liver lysosomal homogenate leading to the formation of an oxime bond-linked daunorubicin-Lys as the smallest active metabolite. In the presence of α-chymotrypsin, all compounds were digested, the degradation rate strongly depending on the type of fatty acid. The bioconjugate containing Lys(nBu) in position 4 was taken up most efficiently by the cancer cells and exerted higher in vitro cytostatic effect than the previously developed GnRH-III((4)Lys(Ac), (8)Lys(Dau = Aoa)) or the parent GnRH-III(Dau = Aoa) bioconjugate. Our results could be explained by the increased binding affinity of the newly developed compound containing Lys(nBu) to the GnRH receptors.