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1.
J Labelled Comp Radiopharm ; 59(4): 164-70, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26968868

RESUMO

Daclatasvir is a novel hepatitis C virus NS5A inhibitor developed by Bristol-Myers Squibb and marketed as Daklinza®. The need to support the development of daclatasvir required the synthesis of carbon-14 labeled material for use in human absorption, distribution, metabolism, and excretion studies. A total of 7.53 mCi of [(14) C]-daclatasvir was synthesized in eight steps from commercially available [(14) C]-copper cyanide. The radiochemical purity was 99.6%, and specific activity was 3.86 µCi/mg. To support a human absolute bioavailability study, 5.56 g of [(13) C2 , (15) N4 ]-daclatasvir was synthesized in four steps.


Assuntos
Imidazóis/síntese química , Imidazóis/farmacocinética , Disponibilidade Biológica , Carbamatos , Técnicas de Química Sintética , Humanos , Imidazóis/química , Imidazóis/metabolismo , Marcação por Isótopo , Pirrolidinas , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Teprotida/síntese química , Teprotida/química , Teprotida/metabolismo , Teprotida/farmacocinética , Valina/análogos & derivados , Proteínas não Estruturais Virais/antagonistas & inibidores
2.
Reprod Domest Anim ; 50(6): 1047-53, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26514336

RESUMO

In the female reproductive tract, the spermatozoa undergo a series of physiological and biochemical changes, prior to gaining the ability to fertilize, that result to capacitation. However, the actin polymerization and protein tyrosine phosphorylation are the two necessary steps for capacitation. In this study, we have demonstrated the actin polymerization and established the correlation between protein tyrosine phosphorylation and actin reorganization during in vitro capacitation in buffalo (Bubalus bubalis) spermatozoa. Indirect immunofluorescence and Western blot techniques were used to detect actin polymerization and tyrosine phosphorylation. The time-dependent fluorimetric studies revealed that the actin polymerization starts from the tail region and progressed towards the head region of spermatozoa during capacitation. The lysophosphatidyl choline (LPC)-induced acrosome reaction (AR) stimulated quick actin depolymerization. The inhibitor cytochalasin D (CD) blocked the in vitro capacitation by inhibiting the actin polymerization. In addition, we also performed different inhibitor (Genistein, H-89, PD9809 and GF-109) and enhancer (dbcAMP, H(2)O(2) and vanadate) studies on actin tyrosine phosphorylation and actin polymerization. The inhibitors of tyrosine phosphorylation inhibit actin tyrosine phosphorylation and polymerization, whereas enhancers of tyrosine phosphorylation stimulate F-actin formation and tyrosine phosphorylation. These observations suggest that the tyrosine phosphorylation regulates the actin polymerization, and both are coupled processes during capacitation of buffalo spermatozoa.


Assuntos
Reação Acrossômica/fisiologia , Actinas/metabolismo , Búfalos/fisiologia , Capacitação Espermática/fisiologia , Espermatozoides/fisiologia , Tirosina/metabolismo , Animais , Bucladesina/química , Peróxido de Hidrogênio/química , Isoquinolinas/química , Masculino , Fosforilação , Polimerização , Sulfonamidas/química , Teprotida/análogos & derivados , Teprotida/química
3.
Biochem Pharmacol ; 96(3): 202-15, 2015 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-26047849

RESUMO

Bradykinin-potentiating peptides (BPPs) from the South American pit viper snake venom were the first natural inhibitors of the human angiotensin I-converting enzyme (ACE) described. The pioneer characterization of the BPPs precursor from the snake venom glands by our group showed for the first time the presence of the C-type natriuretic peptide (CNP) in this same viper precursor protein. The confirmation of the BPP/CNP expression in snake brain regions correlated with neuroendocrine functions stimulated us to pursue the physiological correlates of these vasoactive peptides in mammals. Notably, several snake toxins were shown to have endogenous physiological correlates in mammals. In the present work, we expressed in bacteria the BPPs domain of the snake venom gland precursor protein, and this purified recombinant protein was used to raise specific polyclonal anti-BPPs antibodies. The correspondent single protein band immune-recognized in adult rat brain cytosol was isolated by 2D-SDS/PAGE and/or HPLC, before characterization by MS fingerprint analysis, which identified this protein as superoxide dismutase (SOD, EC 1.15.1.1), a classically known enzyme with antioxidant activity and important roles in the blood pressure modulation. In silico analysis showed the exposition of the BPP-like peptide sequences on the surface of the 3D structure of rat SOD. These peptides were chemically synthesized to show the BPP-like biological activities in ex vivo and in vivo pharmacological bioassays. Taken together, our data suggest that SOD protein have the potential to be a source for putative BPP-like bioactive peptides, which once released may contribute to the blood pressure control in mammals.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Anti-Hipertensivos/química , Hipertensão/tratamento farmacológico , Precursores de Proteínas/química , Superóxido Dismutase/química , Teprotida/química , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anticorpos/química , Anti-Hipertensivos/metabolismo , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Bothrops , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cobaias , Frequência Cardíaca/efeitos dos fármacos , Hipertensão/genética , Hipertensão/metabolismo , Hipertensão/patologia , Masculino , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeo Natriurético Tipo C/química , Peptídeo Natriurético Tipo C/metabolismo , Peptídeo Natriurético Tipo C/farmacologia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Precursores de Proteínas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase/farmacologia , Teprotida/metabolismo , Teprotida/farmacologia
4.
Carbohydr Res ; 346(17): 2688-92, 2011 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22036121

RESUMO

In humans, both the N-terminal catalytic domain (NtMGAM) and the C-terminal catalytic domain (CtMGAM) of small intestinal maltase glucoamylase (MGAM) are α-glycosidases that catalyze the hydrolysis of α-(1→4) glycosidic linkages in the process of starch digestion, and are considered to be the main therapeutic targets for type 2 diabetes. In this work, recombinant human CtMGAM has been cloned for the first time, and this, combined with the expression of NtMGAM in Pichia pastoris, made it possible for us to study the catalytic mechanism of MGAM in a well-defined system. The enzymatic kinetic assays of the two catalytic domains suggest that CtMGAM has the higher affinity for longer maltose oligosaccharides. Kinetic studies of commercially-available drugs such as 1-deoxynojirimycin (DNJ), miglitol, voglibose, and acarbose along with a series of acarviosine-containing oligosaccharides we isolated from Streptomyces coelicoflavus against NtMGAM, CtMGAM, and human pancreatic α-amylase (HPA) provide us an overall profile of the inhibitory ability of these inhibitors. Of all the inhibitors used in this paper, DNJ was the most effective inhibitor against MGAM; the K(i) values for the two catalytic domains were 1.41 and 2.04 µM for NtMGAM and CtMGAM, respectively. Acarviostatins 2-03 and 3-03 were the best inhibitors against HPA with relatively high inhibitory activity against CtMGAM. The acarviostatins 2-03 and 3-03 inhibition constants, K(i), for HPA were 15 and 14.3 nM, and those for CtMGAM were 6.02 and 6.08 µM, respectively. These results suggest that NtMGAM and CtMGAM differ in their substrate specificities and inhibitor tolerance despite their structural relationship.


Assuntos
1-Desoxinojirimicina/química , Inibidores de Glicosídeo Hidrolases , Teprotida/química , 1-Desoxinojirimicina/análogos & derivados , Domínio Catalítico , Humanos , Cinética , alfa-Amilases Pancreáticas/antagonistas & inibidores , alfa-Amilases Pancreáticas/química , Pichia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Especificidade por Substrato , alfa-Glucosidases/biossíntese , alfa-Glucosidases/química
5.
Bioorg Med Chem Lett ; 21(11): 3307-12, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21530250

RESUMO

Ibudilast [1-(2-isopropylpyrazolo[1,5-a]pyridin-3-yl)-2-methylpropan-1-one] is a nonselective phosphodiesterase inhibitor used clinically to treat asthma. Efforts to selectively develop the PDE3- and PDE4-inhibitory activity of ibudilast led to replacement of the isopropyl ketone by a pyridazinone heterocycle. Structure-activity relationship exploration in the resulting 6-(pyrazolo[1,5-a]pyridin-3-yl)pyridazin-3(2H)-ones revealed that the pyridazinone lactam functionality is a critical determinant for PDE3-inhibitory activity, with the nitrogen preferably unsubstituted. PDE4 inhibition is strongly promoted by introduction of a hydrophobic substituent at the pyridazinone N(2) centre and a methoxy group at C-7' in the pyrazolopyridine. Migration of the pyridazinone ring connection from the pyrazolopyridine 3'-centre to C-4' strongly enhances PDE4 inhibition. These studies establish a basis for development of potent PDE4-selective and dual PDE3/4-selective inhibitors derived from ibudilast.


Assuntos
Inibidores de Fosfodiesterase/química , Pirazóis/química , Piridazinas/química , Piridinas/química , Teprotida , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Inibidores de Fosfodiesterase/farmacologia , Pirazóis/farmacologia , Piridazinas/farmacologia , Piridinas/farmacologia , Relação Estrutura-Atividade , Especificidade por Substrato , Teprotida/síntese química , Teprotida/química , Teprotida/farmacologia
6.
Bioorg Med Chem Lett ; 21(10): 2945-8, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21511472

RESUMO

The action of ß-secretase is strongly tied to the onset of Alzheimer's disease. The development of inhibitors of ß-secretase is thus critical to combating this disease, which threatens an ever increasing number of the population and grows in importance as the population ages. Herein we show that flavones from Morus lhou potently inhibit ß-secretase. Our aim in this manuscript is to explore the inhibitory kinetics of natural compounds and develop a phamacophore model which details the critical features responsible for inhibitory activity. The IC(50) values of compounds for ß-secretase inhibition were determined to range between 3.4 and 146.1 µM. Prenylated flavone 2 (IC(50)=3.4 µM) was 20 times more effective than its parent compound, noratocarpetin 1 (IC(50)=60.6 µM). The stronger activity was related with resorcinol moiety on B-ring and isoprenyl functionality at C-3. Kinetic analysis shows that the four effective compounds (1-4) have a noncompetitive mode of action. The binding affinity of flavones for ß-secretase calculated using in silico docking experiments correlated well with their IC(50) values and noncompetitive inhibition modes.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Flavonas/química , Morus/química , Casca de Planta/química , Caules de Planta/química , Teprotida/química , Sítios de Ligação , Ativação Enzimática/efeitos dos fármacos , Flavonas/farmacologia , Concentração Inibidora 50 , Cinética , Modelos Biológicos , Modelos Moleculares , Estrutura Molecular , Prenilação , Teprotida/farmacologia
7.
Bioorg Med Chem ; 18(9): 3320-34, 2010 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-20346684

RESUMO

Novel alpha-mannosidase inhibitors of the type (2R,3R,4S)-2-({[(1R)-2-hydroxy-1-arylethyl]amino}methyl)pyrrolidine-3,4-diol have been prepared and assayed for their anticancer activities. Compound 30 with the aryl group=4-trifluoromethylbiphenyl inhibits the proliferation of primary cells and cell lines of different origins, irrespective of Bcl-2 expression levels, inducing a G2/Mcell cycle arrest and by modification of genes involved in cell cycle progression and survival.


Assuntos
Neoplasias Hematológicas , Neoplasias , Teprotida , alfa-Manosidase/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Teprotida/síntese química , Teprotida/química , Teprotida/farmacologia
8.
Peptides ; 26(7): 1235-47, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15949642

RESUMO

Synthetic analogues of the bradykinin potentiating nonapeptide BPP9alpha indicate significantly different structural requirements for potentiation of the bradykinin (BK)-induced smooth muscle contraction (GPI) and the inhibition of isolated somatic angiotensin I-converting enzyme (ACE). The results disprove the ACE inhibition as the only single mechanism and also the direct interaction of potentiating peptides with the bradykinin receptors in transfected COS-7 cells as molecular mechanism of potentiation. Our results indicate a stimulation of inositol phosphates (IPn) formation independently from the B2 receptor. Furthermore, the results with La3+ support the role of extracellular Ca2+ and its influx through corresponding channels. The missing effect of calyculin on the GPI disproves the role of phosphatases in the potentiating action. These experimental studies should not only contribute to a better understanding of the potentiating mechanisms but also incorporate a shift in the research towards the immune system, in particular towards the immunocompetent polymorphonuclear leukocytes. The chemotaxis of these cells can be potentiated most likely by exclusive inhibition of the enzymatic degradation of bradykinin. Thus the obtained results give evidence that the potentiation of the bradykinin action can occur by different mechanisms, depending on the system and on the applied potentiating factor.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Bradicinina/farmacologia , Contração Muscular/efeitos dos fármacos , Teprotida/análogos & derivados , Sequência de Aminoácidos , Inibidores da Enzima Conversora de Angiotensina/síntese química , Inibidores da Enzima Conversora de Angiotensina/química , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Sinergismo Farmacológico , Cobaias , Humanos , Fosfatos de Inositol/biossíntese , Dados de Sequência Molecular , Músculo Liso/efeitos dos fármacos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Peptidil Dipeptidase A/efeitos dos fármacos , Fosfoproteínas Fosfatases/antagonistas & inibidores , Receptor B2 da Bradicinina/agonistas , Receptor B2 da Bradicinina/genética , Transdução de Sinais/efeitos dos fármacos , Teprotida/química
9.
Biotechnology (N Y) ; 11(8): 930-2, 1993 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7763916

RESUMO

We have developed a new tobacco mosaic virus (TMV) RNA vector and have used it initially to systemically produce an angiotensin-I-converting enzyme inhibitor peptide (ACEI) in tobacco and tomato plants. This vector incorporates a six base 3' context sequence, which permits readthrough of the stop codon for the TMV 130K protein gene, inserted between the stop codon for the coat protein (CP) gene and ACEI gene. In contrast to previous TMV RNA vectors, the new vector produced both an intact CP and a fused protein consisting of CP and ACEI (CP-ACEI). As a result, the vector could form virus particles and spread systemically from inoculated to non-inoculated leaves. In tomato plants, production of ACEI in fruit was also achieved.


Assuntos
Vetores Genéticos , Plantas Geneticamente Modificadas/metabolismo , Teprotida/metabolismo , Vírus do Mosaico do Tabaco/genética , Sequência de Aminoácidos , Sequência de Bases , Capsídeo/genética , Immunoblotting , Dados de Sequência Molecular , Plantas Geneticamente Modificadas/genética , Plantas Tóxicas , RNA Viral/genética , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/biossíntese , Teprotida/química , Nicotiana/metabolismo
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