RESUMO
Proliferation and survival of prostate cancer cells are driven by the androgen receptor (AR) upon binding to androgen steroid hormones. Manipulating the AR signalling axis is the focus for prostate cancer therapy; thus, it is crucial to understand the role of androgens and AR on extracellular vesicle (EV) secretion and cargo. In this study, we report that plasma-derived circulating vesicles consisting of CD9 and double-positive for CD9 and Prostate Specific Membrane Antigen (PSMA) are increased in patients with advanced metastatic prostate cancer, whereas double positives for CD9 and CD63 small extracellular vesicles (S-EVs) are significantly higher in patients with localised prostate cancer. Androgen manipulation by dihydrotestosterone (DHT) and the clinical antagonist enzalutamide (ENZ) altered the heterogeneity and size of CD9 positive S-EVs in AR expressing prostate cancer cells, while assessment of the total number and protein cargo of total S-EVs was unaltered across different treatment groups. Furthermore, hormone stimulation caused strong and specific effects on the small RNA cargo of S-EVs. A total of 543 small RNAs were found to be regulated by androgens including miR-19-3p and miR-361-5p. Analysis of S-EVs heterogeneity and small RNA cargo may provide clinical utility for prostate cancer and be informative to understand further the mechanism of resistance to androgen targeted therapy in castration-resistant prostate cancer.
Assuntos
Androgênios/farmacologia , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/fisiologia , MicroRNAs/metabolismo , Receptores Androgênicos/fisiologia , Tetraspanina 29/metabolismo , Tetraspanina 30/metabolismo , Antígenos de Neoplasias/metabolismo , Antígenos de Superfície/metabolismo , Benzamidas/metabolismo , Benzamidas/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Di-Hidrotestosterona/farmacologia , Humanos , Calicreínas/metabolismo , Masculino , Nitrilas/metabolismo , Nitrilas/farmacologia , Feniltioidantoína/metabolismo , Feniltioidantoína/farmacologia , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata , Transdução de SinaisRESUMO
The antiandrogen therapeutics apalutamide and darolutamide entered the clinic in 2018 and 2019, respectively, for the treatment of castration-resistant prostate cancer (CRPC). Increased expression of the enzyme aldo-keto reductase 1C3 (AKR1C3) is phenotypic of CRPC. The enzyme acts to circumvent castration by producing potent androgens that drive proliferation. Furthermore, AKR1C3 mediates chemotherapeutic resistance to the standard of care, enzalutamide, a structural analogue of apalutamide. Resistance develops in almost all CRPC patients within three months of beginning treatment. Herein, we report that both apalutamide and the structurally distinct darolutamide induce AKR1C3 expression in in vitro models of prostate cancer and are susceptible to AKR1C3-mediated resistance. This effect is countered by pretreatment with a potent and highly selective AKR1C3 inhibitor, sensitizing high AKR1C3 expressing prostate cancer cell lines to the action of both chemotherapeutics with a concomitant reduction in expression of AKR1C3 and the biomarker prostate-specific antigen.
Assuntos
Membro C3 da Família 1 de alfa-Ceto Redutase/metabolismo , Antagonistas de Androgênios/metabolismo , Antineoplásicos/metabolismo , Inibidores Enzimáticos/metabolismo , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Pirazóis/metabolismo , Tioidantoínas/metabolismo , Membro C3 da Família 1 de alfa-Ceto Redutase/genética , Benzamidas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Descoberta de Drogas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Quimioterapia Combinada , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Nitrilas , Orquiectomia , Feniltioidantoína/análogos & derivados , Feniltioidantoína/metabolismo , Antígeno Prostático Específico/metabolismo , Especificidade por SubstratoRESUMO
A liquid chromatography-mass spectrometry assay was developed and validated for simultaneous quantification of anti-hormonal compounds abiraterone, anastrozole, bicalutamide, Δ(4)-abiraterone (D4A), N-desmethyl enzalutamide, enzalutamide, Z-endoxifen, exemestane and letrozole for the purpose of therapeutic drug monitoring (TDM). Plasma samples were prepared with protein precipitation. Analyses were performed with a triple quadrupole mass spectrometer operating in the positive and negative ion-mode. The validated assay ranges from 2 to 200â¯ng/mL for abiraterone, 0.2-20â¯ng/mL for D4A, 10-200â¯ng/mL for anastrozole and letrozole, 1-20â¯ng/mL for Z-endoxifen, 1.88-37.5â¯ng/mL for exemestane and 1500-30,000â¯ng/mL for enzalutamide, N-desmethyl enzalutamide and bicalutamide. Due to low sensitivity for exemestane, the final extract of exemestane patient samples should be concentrated prior to injection and a larger sample volume should be prepared for exemestane patient samples and QC samples to obtain adequate sensitivity. Furthermore, we observed a batch-dependent stability for abiraterone in plasma at room temperature and therefore samples should be shipped on ice. This newly validated method has been successfully applied for routine TDM of anti-hormonal drugs in cancer patients.
Assuntos
Antineoplásicos Hormonais , Monitoramento de Medicamentos/métodos , Administração Oral , Anastrozol/administração & dosagem , Anastrozol/análise , Androstadienos/administração & dosagem , Androstadienos/análise , Androstenos/administração & dosagem , Androstenos/análise , Anilidas/administração & dosagem , Anilidas/análise , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/análise , Benzamidas , Cromatografia Líquida de Alta Pressão , Humanos , Nitrilas/administração & dosagem , Nitrilas/análise , Feniltioidantoína/administração & dosagem , Feniltioidantoína/análogos & derivados , Feniltioidantoína/análise , Feniltioidantoína/metabolismo , Tamoxifeno/administração & dosagem , Tamoxifeno/análogos & derivados , Tamoxifeno/análise , Espectrometria de Massas em Tandem , Compostos de Tosil/administração & dosagem , Compostos de Tosil/análiseRESUMO
BACKGROUND: Enzalutamide is a potent androgen-signaling receptor inhibitor and is licensed for the treatment of metastatic castration-resistant prostate cancer. N-desmethylenzalutamide is the active metabolite of enzalutamide. A method to quantitate enzalutamide and its active metabolite was developed and validated according to the European Medicine Agency guidelines. METHODS: Enzalutamide and N-desmethylenzalutamide were extracted by protein precipitation, separated on a C18 column with gradient elution and analyzed with tandem quadrupole mass spectrometry in positive ion mode. A stable deuterated isotope (D6-enzalutamide) was used as an internal standard. The method was tested and stability was studied in real-life patients with metastatic castration-resistant prostate cancer patients treated with enzalutamide. RESULTS: The calibration curve covered the range of 500-50,000 ng/mL. Within- and between-day precisions were <8% and accuracies were within 108% for both enzalutamide and N-desmethylenzalutamide. Precisions for lower limit of quantification level were <10% and accuracies within 116% for enzalutamide and N-desmethylenzalutamide. Enzalutamide and N-desmethylenzalutamide stability was proven for 24 hours for whole blood at ambient temperature and 23 days for plasma at both ambient temperature and 2-8°C. Long-term patient plasma stability was shown for 14 months at -40°C. CONCLUSIONS: This bioanalytical method was successfully validated and applied to determine plasma concentrations of enzalutamide and N-desmethylenzalutamide in clinical studies and in routine patient care.
Assuntos
Bioensaio/métodos , Feniltioidantoína/análogos & derivados , Neoplasias de Próstata Resistentes à Castração/sangue , Benzamidas , Calibragem , Europa (Continente) , Estudos de Avaliação como Assunto , Humanos , Masculino , Espectrometria de Massas , Nitrilas , Feniltioidantoína/sangue , Feniltioidantoína/metabolismo , Feniltioidantoína/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Reprodutibilidade dos Testes , TemperaturaRESUMO
Enzalutamide is a second-generation androgen receptor (AR) antagonist for the treatment of metastatic castration-resistant prostate cancer (mCRPC). Unfortunately, AR dysfunction means that resistance to enzalutamide will eventually develop. Thus, novel agents are urgently needed to treat this devastating disease. Triptolide (TPL), a key active compound extracted from the Chinese herb Thunder God Vine (Tripterygium wilfordii Hook F.), possesses anti-cancer activity in human prostate cancer cells. However, the effects of TPL against CRPC cells and the underlying mechanism of any such effect are unknown. In this study, we found that TPL at low dose inhibits the transactivation activity of both full-length and truncated AR without changing their protein levels. Interestingly, TPL inhibits phosphorylation of AR and its CRPC-associated variant AR-V7 at Ser515 through XPB/CDK7. As a result, TPL suppresses the binding of AR to promoter regions in AR target genes along with reduced TFIIH and RNA Pol II recruitment. Moreover, TPL at low dose reduces the viability of prostate cancer cells expressing AR or AR-Vs. Low-dose TPL also shows a synergistic effect with enzalutamide to inhibit CRPC cell survival in vitro, and enhances the anti-cancer effect of enzalutamide on CRPC xenografts with minimal side effects. Taken together, our data demonstrate that TPL targets the transactivation activity of both full-length and truncated ARs. Our results also suggest that TPL is a potential drug for CRPC, and can be used in combination with enzalutamide to treat CRPC.
Assuntos
Antineoplásicos Alquilantes/metabolismo , Diterpenos/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fenantrenos/metabolismo , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Benzamidas , Linhagem Celular Tumoral , Modelos Animais de Doenças , Sinergismo Farmacológico , Compostos de Epóxi/metabolismo , Humanos , Masculino , Camundongos Nus , Nitrilas , Feniltioidantoína/metabolismoRESUMO
BACKGROUND: Abiraterone acetate and enzalutamide are 2 novel drugs for the treatment of metastatic castration-resistant prostate cancer. The metabolism of these drugs is extensive. Major metabolites are N-desmethyl enzalutamide, enzalutamide carboxylic acid, abiraterone N-oxide sulfate, and abiraterone sulfate; of which N-desmethyl enzalutamide is reported to possess antiandrogen capacities. A liquid chromatography-tandem mass spectrometry method for simultaneous quantification of abiraterone, enzalutamide, and the main metabolites has been developed and validated to support therapeutic drug monitoring. METHODS: Human plasma samples of patients treated with abiraterone or enzalutamide were harvested at the clinic and stored at -20°C. Proteins were precipitated by acetonitrile, and the final extract was injected on a Kinetex C18 column and separated with gradient elution. Analytes were detected by liquid chromatography-mass spectrometry (Triple Quad 6500). RESULTS: The method was validated over various linear ranges: 1-100 ng/mL for abiraterone, 5-500 ng/mL for enzalutamide and enzalutamide carboxylic acid, 10-1000 ng/mL for N-desmethyl enzalutamide, 30-3000 ng/mL for abiraterone N-oxide sulfate, and 100-10,000 ng/mL for abiraterone sulfate. Intra-assay and interassay variabilities were within ±15% of the nominal concentrations for quality control samples at medium and high concentrations and within ±20% at the lower limit of quantification, respectively. CONCLUSIONS: The described method for simultaneous determination of abiraterone and enzalutamide was validated successfully and provides a useful tool for therapeutic drug monitoring in patients treated with these agents.
Assuntos
Androstenos/sangue , Androstenos/metabolismo , Cromatografia Líquida/métodos , Feniltioidantoína/análogos & derivados , Plasma/química , Espectrometria de Massas em Tandem/métodos , Benzamidas , Monitoramento de Medicamentos/métodos , Humanos , Nitrilas , Feniltioidantoína/sangue , Feniltioidantoína/metabolismo , Reprodutibilidade dos TestesRESUMO
Elderly patients with cancer may have comorbidities, each requiring additional pharmacologic treatment. Therefore, the occurrence of pharmacokinetic (PK) and pharmacodynamic (PD) interactions is very likely, and the risk of adverse reactions (ADRs), due to the narrow therapeutic window of anticancer drugs, is increased. Drug-drug interactions (DDIs) may occur in prostate cancer patients due to inhibition by abiraterone of liver cytochrome P450 (CYP)-dependent enzymes CYP2C8 and 2D6, which are involved in the metabolism of approximately 25% of all drugs, and induction by enzalutamide of CYP3A4, 2C9 and 2C19, which metabolize up to 50% of medications. Therefore, abiraterone may increase plasma levels of CYP2D6 substrates, including amitriptyline, oxycodone and risperidone, as well as of CYP2C8 substrates including amiodarone and carbamazepine. Since enzalutamide is extensively metabolized by CYP2C8, its plasma levels are likely to be raised if coadministered with strong CYP2C8 inhibitors such as gemfibrozil or pioglitazone. Inducers of CYP2C8 (i.e., rifampin) may reduce the effectiveness of enzalutamide and hence should be avoided. Enzalutamide may decrease plasma levels of CYP3A4, 2C9 and 2C19 substrates including disopiramide, quetiapine, quinidine and warfarin. Growing awareness of the importance of DDIs in cancer patients is now reflected in the variety of web-based sources offering information and guidance. However, the evaluation of the clinical relevance of DDIs is the result of a comprehensive evaluation of many factors, including therapeutic index, amplitude of therapeutic range and presence of comorbidities, requiring a specific expertise in clinical pharmacology.
Assuntos
Acetato de Abiraterona/farmacologia , Antineoplásicos/farmacologia , Sistema Enzimático do Citocromo P-450/efeitos dos fármacos , Feniltioidantoína/análogos & derivados , Prednisona/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Acetato de Abiraterona/metabolismo , Benzamidas , Interações Medicamentosas , Humanos , Masculino , Nitrilas , Feniltioidantoína/metabolismo , Feniltioidantoína/farmacologia , PolimedicaçãoRESUMO
BACKGROUND AND OBJECTIVES: Enzalutamide is an androgen receptor inhibitor that has been approved in several countries. Absorption, distribution, metabolism, and excretion (ADME) data in animals would facilitate understanding of the efficacy and safety profiles of enzalutamide, but little information has been reported in public. The purpose of this study was to clarify the missing ADME profile in animals. METHODS: ADME of 14C-enzalutamide after oral administration as Labrasol solution were investigated in non-fasted male Sprague-Dawley rats and beagle dogs. RESULTS: Plasma concentrations of 14C-enzalutamide peaked in rats and dogs at 6-8 h after a single oral administration. In most tissues, radioactivity concentration peaked at 4 h after administration. Excluding the gastrointestinal tract, tissues with the highest concentration of radioactivity were liver, fat, and adrenal glands. The tissue concentrations of radioactivity declined below the limit of quantitation or <0.89 % of maximum concentration by 168 h post-dose. Two known metabolites (M1 and M2) and at least 15 novel possible metabolites were detected in this study. M1 was the most abundant metabolite in both rats and dogs. Unchanged drug was a minor component in excreta. In intact rats, the mean urinary and fecal excretion of radioactivity accounted for 44.20 and 49.80 % of administered radioactivity, respectively. In intact dogs, mean urinary and fecal excretion was 62.00 and 22.30 % of the administered radioactivity, respectively. CONCLUSIONS: Rapid oral absorption was observed in rats and dogs when 14C-enzalutamide was administered as Labrasol solution. Tissue distribution in rats was clarified. The elimination of enzalutamide is mediated primarily by metabolism. Species differences were observed in excretion route.
Assuntos
Antagonistas de Receptores de Andrógenos/sangue , Antagonistas de Receptores de Andrógenos/urina , Feniltioidantoína/análogos & derivados , Administração Oral , Antagonistas de Receptores de Andrógenos/metabolismo , Animais , Benzamidas , Bile/química , Cães , Relação Dose-Resposta a Droga , Fezes/química , Eliminação Hepatobiliar , Masculino , Taxa de Depuração Metabólica , Nitrilas , Absorção pela Mucosa Oral , Feniltioidantoína/sangue , Feniltioidantoína/metabolismo , Feniltioidantoína/urina , Ratos Sprague-Dawley , Especificidade da Espécie , Distribuição TecidualRESUMO
Prostate cancer (PC) is one of the major causes of male death worldwide and the development of new and more potent anti-PC compounds is a constant requirement. Among the current treatments, (R)-bicalutamide and enzalutamide are non-steroidal androgen receptor antagonist drugs approved also in the case of castration-resistant forms. Both these drugs present a moderate antiproliferative activity and their use is limited due to the development of resistant mutants of their biological target. Insertion of fluorinated and perfluorinated groups in biologically active compounds is a current trend in medicinal chemistry, applied to improve their efficacy and stability profiles. As a means to obtain such effects, different modifications with perfluoro groups were rationally designed on the bicalutamide and enzalutamide structures, leading to the synthesis of a series of new antiproliferative compounds. Several new analogues displayed improved in vitro activity towards four different prostate cancer cell lines, while maintaining full AR antagonism and therefore representing promising leads for further development. Furthermore, a series of molecular modelling studies were performed on the AR antagonist conformation, providing useful insights on potential protein-ligand interactions.
Assuntos
Anilidas/síntese química , Anilidas/farmacologia , Desenho de Fármacos , Nitrilas/síntese química , Nitrilas/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/patologia , Compostos de Tosil/síntese química , Compostos de Tosil/farmacologia , Anilidas/química , Anilidas/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Benzamidas , Células CACO-2 , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Técnicas de Química Sintética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Masculino , Microssomos Hepáticos/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Nitrilas/química , Nitrilas/metabolismo , Permeabilidade , Feniltioidantoína/síntese química , Feniltioidantoína/química , Feniltioidantoína/metabolismo , Feniltioidantoína/farmacologia , Conformação Proteica , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Compostos de Tosil/química , Compostos de Tosil/metabolismoRESUMO
Prostate cancer growth depends on androgens. Synthetic antiandrogens are used in the cancer treatment. However, antiandrogens, such as bicalutamide (BIC), have a mixed agonist/antagonist activity. Here we compare the antiandrogenic capacity of BIC to a new antiandrogen, MDV3100 (MDV) or Enzalutamide™. By reconstitution of a hormone-regulated enhancer in Xenopus oocytes we show that both antagonists trigger the androgen receptor (AR) translocation to the nucleus, albeit with a reduced efficiency for MDV. Once in the nucleus, both AR-antagonist complexes can bind sequence specifically to DNA in vivo. The forkhead box transcription factor A (FoxA1) is a negative prognostic indicator for prostate cancer disease. FoxA1 expression presets the enhancer chromatin and makes the DNA more accessible for AR binding. In this context the BIC-AR antiandrogenic effect is seriously compromised as demonstrated by a significant chromatin remodeling and induction of a robust MMTV transcription whereas the MDV-AR complex displays a more persistent antagonistic character.
Assuntos
Anilidas/farmacologia , Antineoplásicos Hormonais/farmacologia , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Neoplasias/metabolismo , Nitrilas/farmacologia , Drogas Antiandrogênicas não Esteroides/farmacologia , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Compostos de Tosil/farmacologia , Anilidas/efeitos adversos , Anilidas/metabolismo , Animais , Antineoplásicos Hormonais/efeitos adversos , Antineoplásicos Hormonais/metabolismo , Benzamidas , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Feminino , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/antagonistas & inibidores , Fator 3-alfa Nuclear de Hepatócito/genética , Humanos , Masculino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Nitrilas/efeitos adversos , Nitrilas/metabolismo , Drogas Antiandrogênicas não Esteroides/efeitos adversos , Drogas Antiandrogênicas não Esteroides/metabolismo , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Feniltioidantoína/metabolismo , Feniltioidantoína/farmacologia , Próstata/efeitos dos fármacos , Próstata/metabolismo , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Compostos de Tosil/efeitos adversos , Compostos de Tosil/metabolismo , Xenopus laevisRESUMO
Metastatic prostate cancer is treated with drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease called castration-resistant prostate cancer, driven by elevated expression of the androgen receptor. Here we characterize the diarylthiohydantoins RD162 and MDV3100, two compounds optimized from a screen for nonsteroidal antiandrogens that retain activity in the setting of increased androgen receptor expression. Both compounds bind to the androgen receptor with greater relative affinity than the clinically used antiandrogen bicalutamide, reduce the efficiency of its nuclear translocation, and impair both DNA binding to androgen response elements and recruitment of coactivators. RD162 and MDV3100 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.
Assuntos
Antagonistas de Androgênios/uso terapêutico , Antineoplásicos/uso terapêutico , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Antagonistas de Androgênios/metabolismo , Antagonistas de Androgênios/farmacocinética , Antagonistas de Androgênios/farmacologia , Anilidas/metabolismo , Anilidas/farmacologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Benzamidas , Disponibilidade Biológica , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Nitrilas/metabolismo , Nitrilas/farmacologia , Feniltioidantoína/metabolismo , Feniltioidantoína/farmacocinética , Feniltioidantoína/farmacologia , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/patologia , Receptores Androgênicos/química , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Compostos de Tosil/metabolismo , Compostos de Tosil/farmacologia , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoAssuntos
Antineoplásicos/uso terapêutico , Feniltioidantoína/análogos & derivados , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/metabolismo , Animais , Antineoplásicos/metabolismo , Benzamidas , Núcleo Celular/metabolismo , Ensaios Clínicos como Assunto , Resistencia a Medicamentos Antineoplásicos , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Nitrilas , Feniltioidantoína/metabolismo , Feniltioidantoína/uso terapêutico , Neoplasias da Próstata/metabolismoRESUMO
Myo-inositol monophosphate phosphatase (IMPP) is a key enzyme in the phosphoinositide cell-signaling system. This study found that incubating the IMPP from a porcine brain with pyridoxal-5'-phosphate (PLP) resulted in a time-dependent enzymatic inactivation. Spectral evidence showed that the inactivation proceeds via the formation of a Schiff's base with the amino groups of the enzyme. After the sodium borohydride reduction of the inactivated enzyme, it was observed that 1.8 mol phosphopyridoxyl residues per mole of the enzyme dimer were incorporated. The substrate, myo-inositol-1-phosphate, protected the enzyme against inactivation by PLP. After tryptic digestion of the enzyme modified with PLP, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. Amino acid sequencing of the peptide identified a portion of the PLP-binding site as being the region containing the sequence L-Q-V-S-Q-Q-E-D-I-T-X, where X indicates that phenylthiohydantoin amino acid could not be assigned. However, the result of amino acid composition of the peptide indicated that the missing residue could be designated as a phosphopyridoxyl lysine. This suggests that the catalytic function of IMPP is modulated by the binding of PLP to a specific lysyl residue at or near its substrate-binding site of the protein.
Assuntos
Encéfalo/enzimologia , Fosfatos de Inositol/metabolismo , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Fosfato de Piridoxal/farmacologia , Aminoácidos/química , Animais , Sítios de Ligação , Boroidretos/farmacologia , Catálise , Cromatografia Líquida de Alta Pressão , Ativação Enzimática/efeitos dos fármacos , Lisina/metabolismo , Oxirredução , Fragmentos de Peptídeos/metabolismo , Feniltioidantoína/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Fosfato de Piridoxal/metabolismo , Suínos , Fatores de TempoRESUMO
The effect of temperature on selectivity in RPLC method development has been evaluated on polybutadiene-coated zirconia. We find that the influence of temperature on selectivity depends strongly on solute type. For solutes of similar structure such as polyaromatic hydrocarbons, temperature has almost no effect on selectivity; however, for solutes with very different functional groups such as chlorophenols, temperature changes did significantly affect selectivity. We feel that simple mixtures with one dominant retention mechanism-e.g., solvophobic retention-will not be helped appreciably by adjusting temperature. However, in complex mixtures with polar and ionizable solutes, optimization by varying the temperature may well be fruitful.
Assuntos
Aminoácidos/isolamento & purificação , Butadienos/química , Clorofenóis/isolamento & purificação , Nucleosídeos/isolamento & purificação , Feniltioidantoína/metabolismo , Compostos Policíclicos/isolamento & purificação , Polímeros/química , Zircônio/metabolismo , Acrilonitrila/química , Aminoácidos/análise , Clorofenóis/análise , Cromatografia Líquida de Alta Pressão , Elastômeros , Nucleosídeos/análise , Peptídeos/análise , Peptídeos/química , Feniltioidantoína/análise , Compostos Policíclicos/análise , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Temperatura , Água/química , Zircônio/químicaRESUMO
A residue of gamma-N-methylasparagine (gamma-NMA) is found at position beta-72 of many phycobiliproteins. delta-N-Methylglutamine is present in some bacterial ribosomal proteins. gamma-NMA was synthesized by reacting the omega-methyl ester of aspartate with methylamine and delta-N-methylglutamine by reaction of pyroglutamate with methylamine. These derivatives and the omega-methyl esters of aspartate and glutamate were characterized by melting point, by thin-layer chromatography, by amino acid analysis, by NMR spectroscopy, and after conversion to the phenylthiohydantoin (PTH) derivative. The gamma-NMA residues in peptides from allophycocyanin, C-phycocyanin, and B-phycoerythrin were stable under the conditions of automated sequential gas-liquid phase Edman degradation. On HPLC, PTH-gamma-NMA co-eluted with PTH-serine and was accompanied by a minor component eluting just prior to dimethylphenylthiourea. Similar results were obtained on manual derivatization of synthetic gamma-NMA to prepare the PTH derivative. The PTH-delta-N-methylglutamine standard eluted near the position of dimethylphenylthiourea under the usual conditions employed for the identification of PTH-amino acid derivatives in automated protein sequencing.
Assuntos
Asparagina/análise , Glutamina/análise , Sequência de Aminoácidos , Bactérias/análise , Cromatografia Líquida de Alta Pressão , Hidrólise , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metilação , Dados de Sequência Molecular , Feniltioidantoína/metabolismoRESUMO
We have studied the domain structure of the A1 heterogeneous nuclear ribonucleoprotein using both partial proteolysis and photochemical cross-linking to oligodeoxynucleotides. Both the intact A1 protein and its proteolytic fragment, the UP1 protein, can be cleaved by Staphylococcus aureus V-8 protease to produce two polypeptides of 92 amino acids. These two polypeptides correspond to the internal repeat sequence previously noted by us to occur in UP1. The two polypeptides can be purified via single-stranded DNA cellulose chromatography and independently cross-linked to [32P]p(dT)8, indicating that each domain can bind to single-stranded nucleic acids. Purification and sequencing of A1 tryptic peptides that had been cross-linked to oligothymidylic acid revealed that 4 phenylalanine residues, phenylalanines 16, 58, 107, and 149 are the sites of covalent adduct formation, with phenylalanine 16 being the major site of cross-linking. These phenylalanine residues are internally homologous when the repeat sequences in A1 are aligned, that is, phenylalanines 16 and 107 occupy analogous positions in the 91-residue repeat, as do phenylalanines 58 and 149. An examination of the primary structures of a variety of eucaryotic RNA-binding proteins with sequence homology to A1 reveals that the cross-linked phenylalanines in A1 are highly conserved among all of these proteins. Our results provide the first experimental evidence that conserved residues in the 90-amino acid repeating domains shared by A1 and other single-stranded nucleic acid binding-proteins form part of an RNA-binding pocket.
Assuntos
Proteínas de Transporte/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B , Ácidos Nucleicos/metabolismo , Fenilalanina/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida , Ribonucleoproteína Nuclear Heterogênea A1 , Ribonucleoproteínas Nucleares Heterogêneas , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Feniltioidantoína/metabolismo , Fotoquímica , Poli T/metabolismo , Proteínas de Ligação a RNA , Ratos , Serina Endopeptidases/metabolismo , Hormônios do Timo/metabolismo , Tripsina/metabolismoRESUMO
Cyanogen bromide peptides are modified with arginine (free base) at the C-terminal homoserin lactone. This modification makes the peptides more hydrophilic. In that way the degradation is possible up to homoserine and the C-terminal arginine. The phenylthiohydantoin derivatives of homoserine and arginine can be detected in good yields by thin-layer or high performance liquid chromatography. The automatic Edman degradation is investigated under various conditions.