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Background: Cardiomyopathy induced by the chemotherapeutic agents doxorubicin and daunorubicin is a major limiting factor for their application in cancer therapy. Chemotactic drug targeting potentially increases the tumor selectivity of drugs and decreases their cardiotoxicity. Increased expression of gonadotropin-releasing hormone (GnRH) receptors on the surface of tumor cells has been reported. Thus, the attachment of the aforementioned chemotherapeutic drugs to GnRH-based peptides may result in compounds with increased therapeutic efficacy. The objective of the present study was to examine the cytotoxic effect of anticancer drug-GnRH-conjugates against two essential cardiovascular cell types, such as cardiomyocytes and endothelial cells. Sixteen different previously developed GnRH-conjugates containing doxorubicin, daunorubicin and methotrexate were investigated in this study. Their cytotoxicity was determined on primary human cardiac myocytes (HCM) and human umbilical vein endothelial cells (HUVEC) using the xCELLigence SP system, which measures impedance changes caused by adhering cells on golden electrode arrays placed at the bottom of the wells. Slopes of impedance-time curves were calculated and for the quantitative determination of cytotoxicity, the difference to the control was analysed. Results: Doxorubicin and daunorubicin exhibited a cytotoxic effect on both cell types, at the highest concentrations tested. Doxorubicin-based conjugates (AN-152, GnRH-III(Dox-O-glut), GnRH-III(Dox-glut-GFLG) and GnRH-III(Dox=Aoa-GFLG) showed the same cytotoxic effect on cardiomyocytes. Among the daunorubicin-based conjugates, [4Lys(Ac)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-YRRL), {GnRH-III(Dau=Aoa-YRRL-C)}2 and {[4N-MeSer]-GnRH-III(Dau-C)}2 had a significant but decreased cytotoxic effect, while the other conjugates - GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-K(Dau=Aoa)), [4Lys(Dau=Aoa)]-GnRH-III(Dau=Aoa), GnRH-III(Dau=Aoa-GFLG), {GnRH-III(Dau-C)}2 and [4N-MeSer]-GnRH-III(Dau=Aoa) - exerted no cytotoxic effect on cardiomyocytes. Mixed conjugates containing methotrexate and daunorubicin - GnRH-III(Mtx-K(Dau=Aoa)) and [4Lys(Mtx)]-GnRH-III(Dau=Aoa) - showed no cytotoxic effect on cardiomyocytes, as well. Conclusion: Based on these results, anticancer drug-GnRH-based conjugates with no cytotoxic effect on cardiomyocytes were identified. In the future, these compounds could provide a more targeted antitumor therapy with no cardiotoxic adverse effects. Moreover, impedimetric cytotoxicity analysis could be a valuable technique to determine the effect of drugs on cardiomyocytes.
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Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the ß-amyloid(1-42) (Aß) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aß-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aß antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aß(4-10) peptide has been identified as an epitope for the polyclonal anti-Aß(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aß-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aß(4-10) peptides upon affinity binding to a monoclonal anti-Aß(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aß(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aß(1-17) to the monoclonal Aß antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aß-specific antibodies with improved therapeutic efficacy.
Assuntos
Alanina/genética , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/química , Alanina/imunologia , Peptídeos beta-Amiloides/genética , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Humanos , Espectrometria de Massas , MutaçãoRESUMO
Cell proliferation and differentiation in multicellular organisms are partially regulated by signaling from the extracellular matrix. The ability to mimic an extracellular matrix would allow particular cell types to be specifically recognized, which is central to tissue engineering. We present a new functional DNA-based material with cell-adhesion properties. It is generated by using covalently branched DNA as primers in PCR. These primers were functionalized by click chemistry with the cyclic peptide c(RGDfK), a peptide that is known to predominantly bind to αvß3 integrins, which are found on endothelial cells and fibroblasts, for example. As a covalent coating of surfaces, this DNA-based material shows cell-repellent properties in its unfunctionalized state and gains adhesiveness towards specific target cells when functionalized with c(RGDfK). These cells remain viable and can be released under mild conditions by DNaseâ I treatment.
Assuntos
DNA/genética , Matriz Extracelular/genética , Peptídeos/genética , Reação em Cadeia da Polimerase , Linhagem Celular Tumoral , DNA/química , Matriz Extracelular/química , Células HEK293 , Células HeLa , Humanos , Conformação Molecular , Peptídeos/químicaRESUMO
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.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Ácido Butírico/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Daunorrubicina/uso terapêutico , Hormônio Liberador de Gonadotropina/uso terapêutico , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/toxicidade , Ácido Butírico/química , Ácido Butírico/toxicidade , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Daunorrubicina/química , Daunorrubicina/toxicidade , Feminino , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/toxicidade , Células HT29 , Coração/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/uso terapêutico , Ácido Pirrolidonocarboxílico/toxicidade , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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The interaction of coenzyme A (CoA) with mercury ions was investigated using electrospray ionization mass spectrometry and circular dichroism spectroscopy. Our results indicated a 1:1 stoichiometric CoA-Hg complex at physiological pH. Furthermore, the CoA conformation considerably changed in the presence of mercury ions. In addition, a by-product of the reaction, thiocoenzyme A, was identified using mass spectrometry.
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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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Daunorrubicina , Sistemas de Liberação de Medicamentos/métodos , Hormônio Liberador de Gonadotropina , Polietilenoglicóis , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Daunorrubicina/química , Daunorrubicina/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Células MCF-7 , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Ratos , SolubilidadeRESUMO
It is well established that gonadotropin-releasing hormone receptors (GnRH-R) are expressed in different types of cancers, including castration-resistant prostate cancer (CRPC) and mediate the antiproliferative effect of GnRH analogs. Thus, these compounds are employed as targeting moieties to selectively deliver chemotherapeutic agents to cancer cells. GnRH-III, the decapeptide isolated from the sea lamprey brain, has lower potency than GnRH in stimulating gonadotropin secretion, but it exerts antiproliferative effects on many tumors expressing the GnRH-R. GnRH-III-based peptides are considered promising targeting moieties for the preparation of anticancer drug delivery systems. These studies were aimed at i) evaluating the antitumor activity of two cytotoxic oxime bond-linked daunorubicin (Dau)-GnRH-III derivative bioconjugates (Dau-GnRH-III, in which daunorubicin was coupled to the 8Lys in the native form of GnRH-III, and Dau-[4Lys(Ac)]-GnRH-III, in which daunorubicin was attached to the 8Lys of a GnRH-III derivative where 4Ser was replaced by an acetylated lysine) on CRPC cells; and ii) to elucidate the involvement of the classical GnRH-R (type I GnRH-R) in this antitumor activity. Our results demonstrated that both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III were rapidly internalized into DU145 prostate cancer cells and exerted a significant cytostatic effect. Both bioconjugates increased the levels of the active form of caspase-3, indicating the involvement of apoptosis in their antitumor activity. The antiproliferative effect of both Dau-GnRH-III and Dau-[4Lys(Ac)]-GnRH-III was counteracted by the simultaneous treatment of the cells with Antide, an antagonist of the GnRH-R. Moreover, after silencing the type I GnRH-R the antitumor activity of both bioconjugates was completely abolished. These data demonstrate that in CRPC cells, daunorubicin-GnRH-III derivative bioconjugates: i) inhibit tumor cell proliferation, by triggering the apoptosis process; ii) exert their antitumor effect through the activation of the type I GnRH-R expressed on these cells. Cytotoxic-GnRH-III derivative may represent promising targeted chemotherapeutics for the treatment of CRPC patients.
Assuntos
Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Oximas/farmacologia , Neoplasias de Próstata Resistentes à Castração/patologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Receptores LHRH/fisiologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Daunorrubicina/química , Relação Dose-Resposta a Droga , Hormônio Liberador de Gonadotropina/química , Humanos , Masculino , Oximas/química , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologia , Células Tumorais CultivadasRESUMO
It is now well established that hormonal pathways are involved in the development of prostate cancer towards the castration resistant (CRPC) stage and can be effective molecular targets for novel treatment strategies. Most CRPC are sensitive to androgens and this can be due to the intratumoral production of androgens, androgen receptor (AR) amplification/ mutations and epigenetic modifications of AR expression/signaling. Based on these observations, potent agents targeting the AR axis were developed: 1) inhibitors of CYP17 (a key enzyme in the production of androgens), such as abiraterone and orteronel; 2) AR antagonists that bind to AR and impair AR activation, such as enzalutamide and ARN-509. Moreover, gonadotropin-releasing hormone receptors (GnRH-R), associated with a strong antitumor activity, are expressed in CRPC cells, indicating that they might represent an important target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists (i.e., degarelix), cytotoxic GnRH-based bioconjugates, delivering chemotherapeutic drugs to cancer cells expressing the GnRH-R, were developed and reported to exert antitumor effects on CRPC cells; some of them (i.e., AN-152) have already entered clinical trials. This review discusses the most relevant patents and recent observations on the anti-cancer efficacy of novel drugs targeting the AR and the GnRH-R pathways in CRPC.
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Antineoplásicos Hormonais/farmacologia , Hormônios/fisiologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Androgênios/fisiologia , Humanos , Masculino , Patentes como Assunto , Receptores Androgênicos/efeitos dos fármacosRESUMO
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 â= Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoaâ=âaminooxyacetyl). 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. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.
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Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Daunorrubicina/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Hormônio Liberador de Gonadotropina/análogos & derivados , Proteínas de Neoplasias/biossíntese , Animais , Daunorrubicina/síntese química , Daunorrubicina/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Eletroforese em Gel Bidimensional , Hormônio Liberador de Gonadotropina/síntese química , Hormônio Liberador de Gonadotropina/farmacologia , Células HT29 , Humanos , Fígado/enzimologia , Lisossomos/enzimologia , Estrutura Molecular , Proteínas de Neoplasias/genética , RatosRESUMO
Recent studies have suggested a protective role of physiological ß-amyloid autoantibodies (Aß-autoantibodies) in Alzheimer's disease (AD). However, the determination of both free and dissociated Aß-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aß-autoantibodies (intact Aß-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects. Both serum and CSF levels of Aß-IgG immune complexes were found to be significantly higher in AD patients compared to control subjects. Moreover, the levels of Aß-IgG complexes were negatively correlated with the cognitive status across the groups, increasing with declining cognitive test performance of the subjects. Our results suggest a contribution of IgG-type autoantibodies to Aß clearance in vivo and an increased immune response in AD, which may be associated with deficient Aß-IgG removal. These findings may contribute to elucidating the role of Aß-autoantibodies in AD pathophysiology and their potential application in AD diagnosis.
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Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/imunologia , Autoanticorpos/sangue , Autoanticorpos/líquido cefalorraquidiano , Ensaio de Imunoadsorção Enzimática/métodos , Idoso , Especificidade de Anticorpos , Cognição/fisiologia , Epitopos/genética , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/líquido cefalorraquidiano , Masculino , Pessoa de Meia-Idade , Modelos EstatísticosRESUMO
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. 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.
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Citostáticos/farmacologia , Daunorrubicina/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Proliferação de Células/efeitos dos fármacos , Citostáticos/síntese química , Citostáticos/química , Daunorrubicina/química , Dimerização , Relação Dose-Resposta a Droga , Estabilidade Enzimática , Hormônio Liberador de Gonadotropina/química , Humanos , Células MCF-7 , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologia , Ratos , Relação Estrutura-AtividadeRESUMO
Prostate cancer is androgen-dependent in its early stages and androgen deprivation therapy represents the most effective first-line therapeutic approach. However, after an initial remission, prostate cancer progresses towards the castration resistant prostate cancer (CRPC) stage, with increased malignancy and resistance to conventional chemotherapy. Pituitary gonadotropin-releasing hormone receptors (GnRH-Rs) represent the most effective molecular target for the treatment of steroid-dependent prostate cancer. GnRH agonists (through GnRH-Rs desensitization) suppress the pituitary-testicular axis and, therefore, represent the treatment of choice for prostate cancer patients. GnRH-Rs are also expressed in prostate cancer, even when the tumor has reached the CRPC stage, and are endowed with antitumor activity, supporting the notion that they might represent a molecular target for GnRH analog-based therapeutic strategies. In addition to GnRH agonists and antagonists, GnRH-based bioconjugates (cytotoxic GnRH bioconjugates, GnRH-conjugated lytic peptides and GnRH-toxin bioconjugates) have been developed and are now undergoing intensive investigations; some of them (i.e., AN-152, Dox-[d-Lys(6)]-GnRH) have entered clinical trials. The advantage of these treatments is the specific delivery of cytotoxic agents to cancer cells. Interestingly, other isoforms of the peptide have been identified. One of them is GnRH-III, which was isolated from sea lamprey. GnRH-III specifically binds to GnRH-Rs in cancer cells and exerts antiproliferative effects; on the other hand, its endocrine effects at pituitary level are insignificant, supporting its selective antitumor activity. Based on these observations, different cytotoxic GnRH-III bioconjugates have recently been synthesized; preliminary in vitro studies suggest that these compounds might represent a new promising treatment strategy for prostate cancer.
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Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Próstata/tratamento farmacológico , Receptores LHRH/metabolismo , Sequência de Aminoácidos , Humanos , Masculino , Dados de Sequência Molecular , Terapia de Alvo Molecular , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de SinaisRESUMO
ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aß epitope recognized by the variable domain of single chain llama anti-Aß-antibodies, termed Aß-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aß-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aß(17-28), in the mid- to carboxy-terminal domain of Aß, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aß(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aß(1-40) or Aß-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aß-nanobodies and Aß(1-40) and the Aß(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aß-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/imunologia , Camelídeos Americanos/imunologia , Epitopos/química , Epitopos/imunologia , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologia , Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Peptídeo Hidrolases/química , Peptídeo Hidrolases/imunologia , Proteólise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Lamprey gonadotropin-releasing hormone-III (lGnRH-III; Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH2), a native isoform of human GnRH (GnRH-I), was initially isolated from the brain of the sea lamprey (Petromyzon marinus). It is a weak GnRH agonist, which exerts a direct antiproliferative effect on cancer cells and has an insignificant LH and FSH releasing potency in mammals. These features reveal the advantages of lGnRH-III and its derivatives for use in cancer therapy. Here we give an overview of various strategies to increase the antitumor activity of lGnRH-III, such as amino acid replacement, cyclization, dimerization and conjugation to polymers or to chemotherapeutic agents. In vitro and in vivo antitumor activity of lGnRH-III based compounds was demonstrated both on hormone dependent and independent tumors.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Hormônio Liberador de Gonadotropina/química , Ácido Pirrolidonocarboxílico/análogos & derivados , Substituição de Aminoácidos , Animais , Desenho de Fármacos , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Lampreias/metabolismo , Maleatos/química , Maleatos/farmacologia , Petromyzon/metabolismo , Povidona/análogos & derivados , Povidona/química , Povidona/farmacologia , Multimerização Proteica , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologiaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Daunorrubicina/farmacologia , Ácidos Graxos/química , Hormônio Liberador de Gonadotropina/farmacologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Acilação , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Daunorrubicina/síntese química , Daunorrubicina/química , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Hormônio Liberador de Gonadotropina/síntese química , Hormônio Liberador de Gonadotropina/química , Células HT29 , Humanos , Células MCF-7 , Estrutura Molecular , Ácido Pirrolidonocarboxílico/síntese química , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/farmacologia , Receptores LHRH/química , Receptores LHRH/metabolismo , Relação Estrutura-AtividadeRESUMO
Physiological ß-amyloid autoantibodies (Aß-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aß antigen-antibody immune complexes. Based on the epitope specificity of the Aß-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aß-IgG immune complexes and (b) an indirect ELISA for the determination of free Aß-autoantibodies. This methodology was applied to the analysis of serum samples from healthy individuals within the age range of 18 to 89 years. Neuropsychological examination of the participants in this study indicated non-pathological, age-related cognitive decline, revealed especially by tests of visual memory and executive function, as well as speed-related tasks. The ELISA serum determinations showed significantly higher levels of Aß-IgG immune complexes compared to free Aß-autoantibodies, while no correlation with age or cognitive performance of the participants was found.
Assuntos
Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/imunologia , Complexo Antígeno-Anticorpo , Autoanticorpos , Disfunção Cognitiva/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/sangue , Autoanticorpos/sangue , Disfunção Cognitiva/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Epitopos/genética , Feminino , Alemanha , Humanos , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Testes NeuropsicológicosRESUMO
Humanin (HN) is a linear 24-aa peptide recently detected in human Alzheimer's disease (AD) brain. HN specifically inhibits neuronal cell death in vitro induced by ß-amyloid (Aß) peptides and by amyloid precursor protein and its gene mutations in familial AD, thereby representing a potential therapeutic lead structure for AD; however, its molecular mechanism of action is not well understood. We report here the identification of the binding epitopes between HN and Aß(1-40) and characterization of the interaction structure through a molecular modeling study. Wild-type HN and HN-sequence mutations were synthesized by SPPS and the HPLC-purified peptides characterized by MALDI-MS. The interaction epitopes between HN and Aß(1-40) were identified by affinity-MS using proteolytic epitope excision and extraction, followed by elution and mass spectrometric characterization of the affinity-bound peptides. The affinity-MS analyses revealed HN(5-15) as the epitope sequence of HN, whereas Aß(17-28) was identified as the Aß interaction epitope. The epitopes and binding sites were ascertained by ELISA of the complex of HN peptides with immobilized Aß(1-40) and by ELISA with Aß(1-40) and Aß-partial sequences as ligands to immobilized HN. The specificity and affinity of the HN-Aß interaction were characterized by direct ESI-MS of the HN-Aß(1-40) complex and by bioaffinity analysis using a surface acoustic wave biosensor, providing a K(D) of the complex of 610 nm. A molecular dynamics simulation of the HN-Aß(1-40) complex was consistent with the binding specificity and shielding effects of the HN and Aß interaction epitopes. These results indicate a specific strong association of HN and Aß(1-40) polypeptide and provide a molecular basis for understanding the neuroprotective function of HN.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Fármacos Neuroprotetores/química , Doença de Alzheimer/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/síntese química , Modelos Moleculares , Conformação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
Assuntos
Daunorrubicina/química , Daunorrubicina/farmacologia , Portadores de Fármacos/química , Hormônio Liberador de Gonadotropina/química , Metotrexato/química , Metotrexato/farmacologia , Fragmentos de Peptídeos/química , Ácido Pirrolidonocarboxílico/análogos & derivados , Sequência de Aminoácidos , Animais , Antineoplásicos/sangue , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Adesão Celular , Proliferação de Células/efeitos dos fármacos , Daunorrubicina/sangue , Daunorrubicina/metabolismo , Estabilidade de Medicamentos , Humanos , Fígado/citologia , Lisossomos/metabolismo , Metotrexato/sangue , Metotrexato/metabolismo , Ácido Pirrolidonocarboxílico/química , RatosRESUMO
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 [(4)Lys]-GnRH-III (Glp-His-Trp-Lys-His-Asp-Trp-Lys-Pro-Gly-NH(2)) 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-NH(2)) was used as a scaffold; an additional lysine residue was coupled to the ϵ-amino group of (8) Lys 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 with a monofunctional compound, both drug design approaches resulted in multifunctional bioconjugates with increased cytostatic effect.