RESUMO
G-quadruplex (G4) DNA structures are widespread in the human genome and are implicated in biologically important processes such as telomere maintenance, gene regulation, and DNA replication. Guanine-rich sequences with potential to form G4 structures are prevalent in the promoter regions of oncogenes, and G4 sites are now considered as attractive targets for anticancer therapies. However, there are very few reports of small "druglike" optical G4 reporters that are easily accessible through one-step synthesis and that are capable of discriminating between different G4 topologies. Here, we present a small water-soluble light-up fluorescent probe that features a minimalistic amidinocoumarin-based molecular scaffold that selectively targets parallel G4 structures over antiparallel and non-G4 structures. We showed that this biocompatible ligand is able to selectively stabilize the G4 template resulting in slower DNA synthesis. By tracking individual DNA molecules, we demonstrated that the G4-stabilizing ligand perturbs DNA replication in cancer cells, resulting in decreased cell viability. Moreover, the fast-cellular entry of the probe enabled detection of nucleolar G4 structures in living cells. Finally, insights gained from the structure-activity relationships of the probe suggest the basis for the recognition of parallel G4s, opening up new avenues for the design of new biocompatible G4-specific small molecules for G4-driven theranostic applications.
Assuntos
Amidinas/química , Cumarínicos/química , DNA/análise , Corantes Fluorescentes/química , Quadruplex G , Amidinas/síntese química , Amidinas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cumarínicos/síntese química , Cumarínicos/metabolismo , DNA/genética , DNA/metabolismo , Replicação do DNA/efeitos dos fármacos , Desenho de Fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Células HeLa , Humanos , Limite de Detecção , Microscopia Confocal , Microscopia de Fluorescência , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Hydroxycinnamic acids represent a versatile group of dietary plant antioxidants. Oxidation of methyl-p-coumarate (pcm) and methyl caffeate (cm) was previously found to yield potent antitumor metabolites. Here, we report the formation of potentially bioactive products of pcm and cm oxidized with peroxynitrite (ONOO¯), a biologically relevant reactive nitrogen species (RNS), or with α,α'-azodiisobutyramidine dihydrochloride (AAPH) as a chemical model for reactive oxygen species (ROS). A continuous flow system was developed to achieve reproducible in situ ONOO¯ formation. Reaction mixtures were tested for their cytotoxic effect on HeLa, SiHa, MCF-7 and MDA-MB-231 cells. The reaction of pcm with ONOO¯ produced two fragments, an o-nitrophenol derivative, and a new chlorinated compound. Bioactivity-guided isolation from the reaction mixture of cm with AAPH produced two dimerization products, including a dihydrobenzofuran lignan that exerted strong antitumor activity in vitro, and has potent in vivo antimetastatic activity which was previously reported. This compound was also detected from the reaction between cm and ONOO¯. Our results demonstrate the ROS/RNS dependent formation of chemically stable metabolites, including a potent antitumor agent (5), from hydroxycinnamic acids. This suggests that diversity-oriented synthesis using ROS/RNS to obtain oxidized antioxidant metabolite mixtures may serve as a valid natural product-based drug discovery strategy.
Assuntos
Amidinas/química , Antineoplásicos/química , Ácidos Cafeicos/química , Ácido Peroxinitroso/química , Amidinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Antioxidantes/química , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Ácidos Cafeicos/metabolismo , Células HeLa , Humanos , Oxirredução , Ácido Peroxinitroso/metabolismo , Espécies Reativas de Nitrogênio/química , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
Nerve endings with immunoreactivity for the P2X3 purinoreceptor (P2X3) in the rat tracheal mucosa were examined by immunohistochemistry of whole-mount preparations with confocal scanning laser microscopy. P2X3 immunoreactivity was observed in ramified endings distributed in the whole length of the trachea. The myelinated parent axons of P2X3-immunoreactive nerve endings ramified into several branches that extended two-dimensionally in every direction at the interface between the epithelial layer and lamina propria. The axonal branches of P2X3-immunoreactive endings branched off many twigs located just beneath the epithelium, and continued to intraepithelial axon terminals. The axon terminals of P2X3-immunoreactive endings were beaded, rounded, or club-like in shape and terminated between tracheal epithelial cells. Flat axon terminals sometimes partly ensheathed neuroendocrine cells with immunoreactivity for SNAP25 or CGRP. Some axons and axon terminals with P2X3 immunoreactivity were immunoreactive for P2X2, while some terminals were immunoreactive for vGLUT2. Furthermore, a retrograde tracing method using fast blue (FB) revealed that 88.4% of FB-labeled cells with P2X3 immunoreactivity originated from the nodose ganglion. In conclusion, P2X3-immunoreactive nerve endings in the rat tracheal mucosa have unique morphological characteristics, and these endings may be rapidly adapting receptors and/or irritant receptors that are activated by mucosal irritant stimuli.
Assuntos
Mucosa/citologia , Terminações Nervosas/metabolismo , Receptores Purinérgicos P2X3/metabolismo , Células Receptoras Sensoriais/citologia , Traqueia/citologia , Amidinas/metabolismo , Animais , Calcitonina/metabolismo , Células Epiteliais/citologia , Masculino , Microscopia Confocal , Proteína Básica da Mielina/metabolismo , Gânglio Nodoso/citologia , Ratos , Ratos Wistar , Proteína 25 Associada a Sinaptossoma/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
Tyrosine nitration is an oxidative post-translational modification that can occur in proteins associated to hydrophobic bio-structures such as membranes and lipoproteins. In this work, we have studied tyrosine nitration in membranes using a model system consisting of phosphatidylcholine liposomes with pre-incorporated tyrosine-containing 23 amino acid transmembrane peptides. Tyrosine residues were located at positions 4, 8 or 12 of the amino terminal, resulting in different depths in the bilayer. Tyrosine nitration was accomplished by exposure to peroxynitrite and a peroxyl radical donor or hemin in the presence of nitrite. In egg yolk phosphatidylcholine liposomes, nitration was highest for the peptide with tyrosine at position 8 and dramatically increased as a function of oxygen levels. Molecular dynamics studies support that the proximity of the tyrosine phenolic ring to the linoleic acid peroxyl radicals contributes to the efficiency of tyrosine oxidation. In turn, α-tocopherol inhibited both lipid peroxidation and tyrosine nitration. The mechanism of tyrosine nitration involves a "connecting reaction" by which lipid peroxyl radicals oxidize tyrosine to tyrosyl radical and was fully recapitulated by computer-assisted kinetic simulations. Altogether, this work underscores unique characteristics of the tyrosine oxidation and nitration process in lipid-rich milieu that is fueled via the lipid peroxidation process.
Assuntos
Membrana Celular/metabolismo , Radicais Livres/metabolismo , Peroxidação de Lipídeos , Peptídeos/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/metabolismo , Amidinas/metabolismo , Sequência de Aminoácidos , Membrana Celular/química , Hemina/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Simulação de Dinâmica Molecular , Oxirredução , Oxigênio/metabolismo , Peptídeos/química , Tirosina/químicaRESUMO
DB1255 is a symmetrical diamidinophenyl-dithiophene that exhibits cellular activity by binding to DNA and inhibiting binding of ERG, an ETS family transcription factor that is commonly overexpressed or translocated in leukemia and prostate cancer [Nhili, R., Peixoto, P., Depauw, S., Flajollet, S., Dezitter, X., Munde, M. M., Ismail, M. A., Kumar, A., Farahat, A. A., Stephens, C. E., Duterque-Coquillaud, M., Wilson, W. D., Boykin, D. W., and David-Cordonnier, M. H. (2013) Nucleic Acids Res. 41, 125-138]. Because transcription factor inhibition is complex but is an attractive area for anticancer and antiparasitic drug development, we have evaluated the DNA interactions of additional derivatives of DB1255 to gain an improved understanding of the biophysical chemistry of complex function and inhibition. DNase I footprinting, biosensor surface plasmon resonance, and circular dichroism experiments show that DB1255 has an unusual and strong monomer binding mode in minor groove sites that contain a single GC base pair flanked by AT base pairs, for example, 5'-ATGAT-3'. Closely related derivatives, such as compounds with the thiophene replaced with furan or selenophane, bind very weakly to GC-containing sequences and do not have biological activity. DB1255 is selective for the ATGAT site; however, a similar sequence, 5'-ATGAC-3', binds DB1255 more weakly and does not produce a footprint. Molecular docking studies show that the two thiophene sulfur atoms form strong, bifurcated hydrogen bond-type interactions with the G-N-H sequence that extends into the minor groove while the amidines form hydrogen bonds to the flanking AT base pairs. The central dithiophene unit of DB1255 thus forms an excellent, but unexpected, single-GC base pair recognition module in a monomer minor groove complex.
Assuntos
Amidinas/química , DNA/química , Tiofenos/química , Amidinas/síntese química , Amidinas/metabolismo , Sequência de Aminoácidos , Técnicas Biossensoriais , DNA/metabolismo , Pegada de DNA , Desoxirribonuclease I/metabolismo , Guanina/química , Guanina/metabolismo , Modelos Moleculares , Ressonância de Plasmônio de Superfície , Tiofenos/síntese química , Tiofenos/metabolismo , Temperatura de TransiçãoRESUMO
To establish the rates and mechanisms of decomposition of guanidine and amidine derivatives in aqueous solution and the rate enhancements produced by the corresponding enzymes, we examined their rates of reaction at elevated temperatures and used the Arrhenius equation to extrapolate the results to room temperature. The similar reactivities of methylguanidine and 1,1,3,3-tetramethylguanidine and their negative entropies of activation imply that their decomposition proceeds by hydrolysis rather than elimination. The influence of changing pH on the rate of decomposition is consistent with attack by hydroxide ion on the methylguanidinium ion (k2 = 5 × 10(-6) M(-1) s(-1) at 25 °C) or with the kinetically equivalent attack by water on uncharged methylguanidine. At 25 °C and pH 7, N-methylguanidine is several orders of magnitude more stable than acetamidine, urea, or acetamide. Under the same conditions, the enzymes arginase and agmatinase accelerate substrate hydrolysis 4 × 10(14)-fold and 6 × 10(12)-fold, respectively, by mechanisms that appear to involve metal-mediated water attack. Arginine deiminase accelerates substrate hydrolysis 6 × 10(12)-fold by a mechanism that (in contrast to the mechanisms employed by arginase and agmatinase) is believed to involve attack by an active-site cysteine residue.
Assuntos
Amidinas/metabolismo , Guanidina/metabolismo , Amidinas/química , Guanidina/química , Temperatura Alta , Concentração de Íons de Hidrogênio , Hidrólise , Metilguanidina/química , Metilguanidina/metabolismo , Estrutura Molecular , Termodinâmica , Água/químicaRESUMO
Visceral afferents expressing transient receptor potential (TRP) channels TRPV1 and TRPA1 are thought to be required for neurogenic inflammation and development of inflammatory hyperalgesia. Using a mouse model of chronic pancreatitis (CP) produced by repeated episodes (twice weekly) of caerulein-induced AP (AP), we studied the involvement of these TRP channels in pancreatic inflammation and pain-related behaviors. Antagonists of the two TRP channels were administered at different times to block the neurogenic component of AP. Six bouts of AP (over 3 wks) increased pancreatic inflammation and pain-related behaviors, produced fibrosis and sprouting of pancreatic nerve fibers, and increased TRPV1 and TRPA1 gene transcripts and a nociceptive marker, pERK, in pancreas afferent somata. Treatment with TRP antagonists, when initiated before week 3, decreased pancreatic inflammation and pain-related behaviors and also blocked the development of histopathological changes in the pancreas and upregulation of TRPV1, TRPA1, and pERK in pancreatic afferents. Continued treatment with TRP antagonists blocked the development of CP and pain behaviors even when mice were challenged with seven more weeks of twice weekly caerulein. When started after week 3, however, treatment with TRP antagonists was ineffective in blocking the transition from AP to CP and the emergence of pain behaviors. These results suggest: (1) an important role for neurogenic inflammation in pancreatitis and pain-related behaviors, (2) that there is a transition from AP to CP, after which TRP channel antagonism is ineffective, and thus (3) that early intervention with TRP channel antagonists may attenuate the transition to and development of CP effectively.
Assuntos
Oximas/uso terapêutico , Dor/prevenção & controle , Pancreatite Crônica/tratamento farmacológico , Piridinas/uso terapêutico , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Amidinas/metabolismo , Analgésicos Opioides/uso terapêutico , Análise de Variância , Animais , Antígenos de Diferenciação/metabolismo , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Ceruletídeo/toxicidade , Modelos Animais de Doenças , Progressão da Doença , Comportamento Exploratório/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Monócitos/patologia , Morfina/uso terapêutico , Infiltração de Neutrófilos/efeitos dos fármacos , Gânglio Nodoso/metabolismo , Gânglio Nodoso/patologia , Dor/etiologia , Dor/patologia , Medição da Dor/efeitos dos fármacos , Pâncreas/efeitos dos fármacos , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite Crônica/induzido quimicamente , Pancreatite Crônica/complicações , Pancreatite Crônica/patologia , Peroxidase/metabolismo , RNA Mensageiro/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/patologia , Canal de Cátion TRPA1 , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Fatores de Tempo , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Direct modulation of gene expression by targeting oncogenic transcription factors is a new area of research for cancer treatment. ERG, an ETS-family transcription factor, is commonly over-expressed or translocated in leukaemia and prostate carcinoma. In this work, we selected the di-(thiophene-phenyl-amidine) compound DB1255 as an ERG/DNA binding inhibitor using a screening test of synthetic inhibitors of the ERG/DNA interaction followed by electrophoretic mobility shift assays (EMSA) validation. Spectrometry, footprint and biosensor-surface plasmon resonance analyses of the DB1255/DNA interaction evidenced sequence selectivity and groove binding as dimer. Additional EMSA evidenced the precise DNA-binding sequence required for optimal DB1255/DNA binding and thus for an efficient ERG/DNA complex inhibition. We further highlighted the structure activity relationships from comparison with derivatives. In cellulo luciferase assay confirmed this modulation both with the constructed optimal sequences and the Osteopontin promoter known to be regulated by ERG and which ERG-binding site was protected from DNaseI digestion on binding of DB1255. These data showed for the first time the ERG/DNA complex modulation, both in vitro and in cells, by a heterocyclic diamidine that specifically targets a portion of the ERG DNA recognition site.
Assuntos
Amidinas/farmacologia , Antineoplásicos/farmacologia , Tiofenos/farmacologia , Transativadores/antagonistas & inibidores , Ativação Transcricional/efeitos dos fármacos , Amidinas/química , Amidinas/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA/química , DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Tiofenos/química , Tiofenos/metabolismo , Transativadores/metabolismo , Regulador Transcricional ERGRESUMO
Caffeic, syringic, and protocatechuic acids are phenolic acids derived directly from food intake or come from the gut metabolism of polyphenols. In this study, the antioxidant activity of these compounds was at first evaluated in membrane models, where caffeic acid behaved as a very effective chain-breaking antioxidant, whereas syringic and protocatechuic acids were only retardants of lipid peroxidation. However, all three compounds acted as good scavengers of reactive species in cultured cells subjected to exogenous oxidative stress produced by low level of H(2)O(2). Many tumour cells are characterised by increased ROS levels compared with their noncancerous counterparts. Therefore, we investigated whether phenolic acids, at low concentrations, comparable to those present in human plasma, were able to decrease basal reactive species. Results show that phenolic acids reduced ROS in a leukaemia cell line (HEL), whereas no effect was observed in normal cells, such as HUVEC. The compounds exhibited no toxicity to normal cells while they decreased proliferation in leukaemia cells, inducing apoptosis. In the debate on optimal ROS-manipulating strategies in cancer therapy, our work in leukaemia cells supports the antioxidant ROS-depleting approach.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Dieta , Hidroxibenzoatos/farmacologia , Leucemia/patologia , Membranas Artificiais , Modelos Biológicos , Amidinas/metabolismo , Antioxidantes/química , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Hidroxibenzoatos/química , Leucemia/enzimologia , Peroxidação de Lipídeos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxigênio/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
Motor neurons (MNs) are designated as alpha/gamma and fast/slow based on their target sites and the types of muscle fibers innervated; however, few molecular markers that distinguish between these subtypes are available. Here we report that osteopontin (OPN) is a selective marker of alpha MNs in the mouse spinal cord. OPN was detected in approximately 70% of postnatal choline acetyltransferase (ChAT)-positive MNs with relatively large somas, but not in those with smaller somas. OPN+/ChAT+ MNs were also positive for NeuN, an alpha MN marker, but were negative for Err3, a gamma MN marker. The size distribution of OPN+/ChAT+ cells was nearly identical to that of NeuN+/ChAT+ alpha MNs. Group Ia proprioceptive terminals immunoreactive for vesicular glutamate transporter-1 were selectively detected on the OPN+/ChAT+ cells. OPN staining was also detected at motor axon terminals at neuromuscular junctions, where the OPN+ terminals were positive or negative for SV2A, a marker distinguishing fast/slow motor endplates. Finally, retrograde labeling following intramuscular injection of fast blue indicated that OPN is expressed in both fast and slow MNs. Collectively, our findings show that OPN is an alpha MN marker present in both the soma and the endplates of alpha MNs in the postnatal mouse spinal cord.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios Motores/metabolismo , Osteopontina/metabolismo , Medula Espinal/citologia , Amidinas/metabolismo , Animais , Animais Recém-Nascidos , Bungarotoxinas/farmacocinética , Contagem de Células , Colina O-Acetiltransferase/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/inervação , Músculos/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Osteopontina/deficiência , Fosfopiruvato Hidratase/metabolismo , Células do Corno Posterior/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Receptores de Estrogênio/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/metabolismoRESUMO
A combination of biophysical, biochemical, and computational techniques was used to delineate mechanistic differences between the platinum-acridine hybrid agent [PtCl(en)(L)](NO(3))(2) (complex 1, en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) and a considerably more potent second-generation analogue containing L' = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine (complex 2). Calculations at the density functional theory level provide a rationale for the binding preference of both complexes for guanine-N7 and the relatively high level of adenine adducts observed for compound 1. A significant rate enhancement is observed for binding of the amidine-based complex 2 with DNA compared with the thiourea-based prototype 1. Studies conducted with chemical probes and on the bending and unwinding of model duplex DNA suggest that adducts of complex 2 perturb B-form DNA more severely than complex 1, however, without denaturing the double strand and significantly less than cisplatin. Circular and linear dichroism spectroscopies and viscosity measurements suggest that subtle differences exist between the intercalation modes and adduct geometries of the two complexes. The adducts formed by complex 2 most efficiently inhibit transcription of the damaged DNA by RNA polymerase II. Not only do complexes 1 and 2 cause less distortion to DNA than cisplatin, they also do not compromise the thermodynamic stability of the modified duplex. This leads to a decreased or negligible affinity of HMG domain proteins for the adducts formed by either Pt-acridine complex. In a DNA repair synthesis assay the lesions formed by complex 2 were repaired less efficiently than those formed by complex 1. These significant differences in DNA adduct formation, structure, and recognition between the two acridine complexes and cisplatin help to elucidate why compound 2 is highly active in cisplatin-resistant, repair proficient cancer cell lines.
Assuntos
Acridinas/química , Amidinas/química , Antineoplásicos/química , Adutos de DNA/química , Reparo do DNA/efeitos dos fármacos , DNA/metabolismo , Compostos Organoplatínicos/química , Acridinas/metabolismo , Acridinas/farmacologia , Amidinas/metabolismo , Amidinas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Cisplatino/análogos & derivados , Cisplatino/química , Cisplatino/metabolismo , Cisplatino/farmacologia , DNA/química , DNA de Forma B/química , DNA de Forma B/metabolismo , Desenho de Fármacos , Proteína HMGB1/metabolismo , Células HeLa , Humanos , Substâncias Intercalantes/química , Substâncias Intercalantes/metabolismo , Substâncias Intercalantes/farmacologia , Cinética , Conformação de Ácido Nucleico/efeitos dos fármacos , Compostos Organoplatínicos/metabolismo , Compostos Organoplatínicos/farmacologia , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade , Tioureia/química , Tioureia/metabolismo , Tioureia/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
The primary motor cortex (M1) of the rat contains dopaminergic terminals. The origin of this dopaminergic projection and its functional role for movement are obscure. Other areas of cortex receive dopaminergic projections from the ventral tegmental area (VTA) of the midbrain, and these projections are involved in learning phenomena. We therefore hypothesized that M1 receives a dopaminergic projection from VTA and that this projection mediates the learning of a motor skill by inducing cellular plasticity events in M1. Retrograde tracing from M1 of Long-Evans rats in conjunction with tyrosine hydroxylase immunohistochemistry identified dopaminergic cell bodies in VTA. Electrical stimulation of VTA induced expression of the immediate-early gene c-fos in M1, which was blocked by intracortical injections of D(1) and D(2) antagonists. Destroying VTA dopaminergic neurons prevented the improvements in forelimb reaching seen in controls during daily training. Learning recovered on administration of levodopa into the M1 of VTA-lesioned animals. Lesioning VTA did not affect performance of an already learned skill, hence, left movement execution intact. These findings provide evidence that dopaminergic terminals in M1 originate in VTA, contribute to M1 plasticity, and are necessary for successful motor skill learning. Because VTA dopaminergic neurons are known to signal rewards, the VTA-to-M1 projection is a candidate for relaying reward information that could directly support the encoding of a motor skill within M1.
Assuntos
Condicionamento Operante/fisiologia , Dopamina/metabolismo , Córtex Motor/fisiologia , Destreza Motora/fisiologia , Neurônios/fisiologia , Área Tegmentar Ventral/citologia , Adrenérgicos/toxicidade , Vias Aferentes/efeitos dos fármacos , Vias Aferentes/fisiologia , Amidinas/metabolismo , Animais , Comportamento Animal/fisiologia , Benzazepinas/farmacologia , Condicionamento Operante/efeitos dos fármacos , Antagonistas de Dopamina/farmacologia , Combinação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Masculino , Córtex Motor/efeitos dos fármacos , Oxidopamina/toxicidade , Proteínas Proto-Oncogênicas c-fos/metabolismo , Racloprida/farmacologia , Ratos , Ratos Long-Evans , Tempo de Reação/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Área Tegmentar Ventral/lesões , Área Tegmentar Ventral/fisiologiaRESUMO
Cinnamoylphenethylamine (CNPA) derivatives including feruloylphenethylamine (FRPA), caffeoylphenethylamine (CFPA), cinnamoyltyramine (CNTA), feruloyltyramine (FRTA) and caffeoyltyramine (CFTA) were synthesized in order to investigate the influence of the number and position of hydroxyl group on Cu(2+)/glutathione (GSH) and 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH)-induced oxidation of DNA. The radical-scavenging properties of these CNPA derivatives were also evaluated by trapping 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonate) cationic radical (ABTS(+â¢)), 2,2'-diphenyl-1-picrylhydrazyl radical (DPPH) and galvinoxyl radical. In addition, these CNPA derivatives were tested by linoleic acid (LH)-ß-carotene-bleaching experiment. The chemical kinetic was employed to treat the results from AAPH-induced oxidation of DNA and gave the order of antioxidant ability as CFTA > CFPA > FRTA > FRPA. CFTA and CFPA also possessed high abilities to inhibit Cu²(+)/GSH-mediated degradation of DNA, whereas FRPA and FRTA can protect LH against the auto-oxidation efficiently. Finally, CFPA and FRPA exhibited high activity in trapping ABTS(+â¢), DPPH and galvinoxyl radicals. Therefore, the cinnamoyl group bearing ortho-dihydroxyl or hydroxyl with ortho-methoxyl benefited for CNPA derivatives to protect DNA, while hydroxyl in tyramine cannot enhance the radical-scavenging abilities of CNPA derivatives.
Assuntos
Antioxidantes/metabolismo , Cinamatos/metabolismo , DNA/metabolismo , Sequestradores de Radicais Livres/metabolismo , Fenetilaminas/metabolismo , Amidinas/metabolismo , Antioxidantes/síntese química , Compostos Benzidrílicos/metabolismo , Benzotiazóis/metabolismo , Compostos de Bifenilo/metabolismo , Cinamatos/síntese química , Cobre/química , Cobre/metabolismo , Sequestradores de Radicais Livres/síntese química , Glutationa/metabolismo , Hidróxidos/metabolismo , Ácido Linoleico/metabolismo , Oxirredução/efeitos dos fármacos , Fenetilaminas/síntese química , Picratos/metabolismo , Soluções/química , Soluções/metabolismo , Relação Estrutura-Atividade , Ácidos Sulfônicos/metabolismo , beta Caroteno/metabolismoRESUMO
Matriptase is a serine protease expressed by several types of cancer cells and it participates in tumour growth and progression through the activation of hepatocyte growth factor (HGF) and urokinase-type plasminogen activator (uPA). The metabolism of two potent and selective peptidomimetic inhibitors of matriptase (CJ-1737 and CJ-672) was examined in vitro with enzyme preparations (9000g supernatants, microsomes, and plasma) from dog, pig, rat, and human. It was found that both compounds displayed interesting species-dependent differences. Though CJ-1737 was not metabolized by microsomes, by 9000g supernatants from all species, or by human or rat plasma, canine and porcine plasma enzymes rapidly hydrolysed this compound. In contrast, CJ-672 was metabolized exclusively by enzymes from human liver (microsomes and 9000g supernatants) via a two-step metabolic pathway. Additionally, the distribution of both compounds was investigated in mice. The highest amounts were measured in the kidney and liver, followed by the spleen, lung, and heart. In contrast to CJ-1737, high concentrations of CJ-672 were detected in the colon, indicating an additional biliary excretion. In summary, this work clarifies both the metabolism and distribution of two new matriptase inhibitors and demonstrates important metabolic differences between human enzymes and those from commonly used laboratory animals.
Assuntos
Amidinas/metabolismo , Inibidores Enzimáticos/metabolismo , Fenilalanina/análogos & derivados , Amidinas/administração & dosagem , Amidinas/farmacocinética , Animais , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Colo/metabolismo , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Injeções Intraperitoneais , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Espectrometria de Massas , Camundongos , Miocárdio/metabolismo , Fenilalanina/administração & dosagem , Fenilalanina/metabolismo , Fenilalanina/farmacocinética , Ratos , Serina Endopeptidases , Especificidade da Espécie , Baço/metabolismo , SuínosRESUMO
Liver and bile secretion can be an important first-pass and clearance route for drug compounds and also the site of several drug-drug interactions. In the clinical program for ximelagatran development, an unexpected effect of erythromycin on the pharmacokinetics of the direct thrombin inhibitor ximelagatran and its metabolites was detected. This interaction was believed to be mediated by inhibition of drug transporters, which normally extrude the drug into the bile. Previous Caco-2 cell experiments indicated the involvement of an active efflux mechanism for ximelagatran, hydroxy-melagatran, and melagatran possibly mediated by P-glycoprotein (P-gp). However, the inhibitors used may not have been specific enough and the possibility that transporters other than P-gp were important in the Caco-2 cell assay cannot be excluded. In this study we used RNA interference, a post-transcriptional gene silencing mechanism in which mRNA is degraded in a sequence-specific manner, to specifically knock down P-gp or multidrug resistance-associated protein 2 (MRP2) transporters in Caco-2 cells. The data obtained from bidirectional transport studies in these cells indicate a clear involvement of P-gp but not of MRP2 in the transport of ximelagatran, hydroxy-melagatran, and melagatran across the apical cell membrane. The present study shows that short hairpin RNA Caco-2 cells are a valuable tool to investigate the contribution of specific transporters in the transcellular transport of drug molecules and to predict potential sites of pharmacokinetic interactions. The results also suggest that inhibition of hepatic P-gp is involved in the erythromycin-ximelagatran interaction seen in clinical studies.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Anticoagulantes/farmacocinética , Azetidinas/farmacocinética , Benzilaminas/farmacocinética , Técnicas de Silenciamento de Genes , Sequências Repetidas Invertidas , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Trombina/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Amidinas/metabolismo , Azetidinas/metabolismo , Benzilaminas/metabolismo , Biotransformação , Células CACO-2 , Polaridade Celular , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica , Humanos , Lentivirus , Moduladores de Transporte de Membrana/farmacocinética , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismoRESUMO
Molecules that block nitric oxide's (NO) biosynthesis are of significant interest. For example, nitric oxide synthase (NOS) inhibitors have been suggested as antitumor therapeutics, as have inhibitors of dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that catabolizes endogenous NOS inhibitors. Dual-targeted inhibitors hold promise as more effective reagents to block NO biosynthesis than single-targeted compounds. In this study, a small set of known NOS inhibitors are surveyed as inhibitors of recombinant human DDAH-1. From these, an alkylamidine scaffold is selected for homologation. Stepwise lengthening of one substituent converts an NOS-selective inhibitor into a dual-targeted NOS/DDAH-1 inhibitor and then into a DDAH-1 selective inhibitor, as seen in the inhibition constants of N5-(1-iminoethyl)-, N5-(1-iminopropyl)-, N5-(1-iminopentyl)- and N(5)-(1-iminohexyl)-l-ornithine for neuronal NOS (1.7, 3, 20, >1,900 microM, respectively) and DDAH-1 (990, 52, 7.5, 110 microM, respectively). A 1.9 A X-ray crystal structure of the N5-(1-iminopropyl)-L-ornithine:DDAH-1 complex indicates covalent bond formation between the inhibitor's amidino carbon and the active-site Cys274, and solution studies show reversible competitive inhibition, consistent with a reversible covalent mode of DDAH inhibition by alkylamidine inhibitors. These represent a versatile scaffold for the development of a targeted polypharmacological approach to control NO biosynthesis.
Assuntos
Amidoidrolases/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/síntese química , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico/antagonistas & inibidores , Amidinas/metabolismo , Amidinas/farmacologia , Amidoidrolases/metabolismo , Cristalografia por Raios X , Sistemas de Liberação de Medicamentos/métodos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Humanos , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo I/metabolismo , Ornitina/análogos & derivados , Ornitina/metabolismo , Ornitina/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Tioureia/metabolismo , Tioureia/farmacologiaRESUMO
We investigated the effects of two flavonoids quercetin and dihydroquercetin (DHQ), which have different solubilities and antioxidant capacities, on hemolysis and platelet aggregation in human blood. Exposure of human red blood cells (RBCs) to free radicals generated by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) for 2h resulted in 63.5+/-3.9% hemolysis (vehicle: 0.3+/-0.4%). Pre-incubation of RBCs with lipid-soluble quercetin and water-soluble DHQ for 30min significantly reduced the AAPH-induced hemolysis to 3.6+/-1.5% and 32.5+/-5.6% respectively. In contrast, quercetin and DHQ were similarly effective in reducing phospholipase C-induced hemolysis (37.2+/-9.1% and 45.4+/-10.0% versus vehicle 75.7+/-5.2%, P<0.001). Pre-incubation with quercetin, but not DHQ, inhibited the aggregation of platelets by adenosine diphosphate. DHQ was more potent than quercetin in inhibiting superoxide produced by xanthine oxidase. These results suggest that the antihemolytic effects of flavonoids may not be directly mediated by removal of free radicals and may likely be due to their interaction with cell membrane.
Assuntos
Antioxidantes/farmacologia , Plaquetas/efeitos dos fármacos , Membrana Eritrocítica/efeitos dos fármacos , Quercetina/análogos & derivados , Quercetina/farmacologia , Difosfato de Adenosina/metabolismo , Amidinas/metabolismo , Plaquetas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Eritrocítica/metabolismo , Hemólise/fisiologia , Humanos , Oxidantes/metabolismo , Agregação Plaquetária/efeitos dos fármacos , Superóxidos/metabolismo , Fosfolipases Tipo C/metabolismoRESUMO
A hydroxyamidine chemotype has been discovered as a key pharmacophore in novel inhibitors of indoleamine 2,3-dioxygenase (IDO). Optimization led to the identification of 5l, which is a potent (HeLa IC(50) = 19 nM) competitive inhibitor of IDO. Testing of 5l in mice demonstrated pharmacodynamic inhibition of IDO, as measured by decreased kynurenine levels (>50%) in plasma and dose dependent efficacy in mice bearing GM-CSF-secreting B16 melanoma tumors.
Assuntos
Ligação Competitiva , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Melanoma/enzimologia , Amidinas/química , Amidinas/metabolismo , Amidinas/farmacologia , Amidinas/uso terapêutico , Animais , Modelos Animais de Doenças , Progressão da Doença , Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/química , Concentração Inibidora 50 , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Camundongos , Modelos Moleculares , Conformação MolecularRESUMO
BACKGROUND: Melatonin is well-established as a powerful reducing agent of oxidant generated in the cell medium. We aimed to investigate how readily melatonin is oxidized by peroxyl radicals ROO generated by the thermolysis of 2,2'-azobis(2-amidinopropane) hydrochloride (AAPH) and the role of glutathione (GSH) during the reaction course. METHODS: Chromatographic, mass spectroscopy, and UV-visible spectrometric techniques were used to study the oxidation of melatonin by ROO or horseradish peroxidase (HRP)/H2O2. Our focus was the characterization of products and the study of features of the reaction. RESULTS: We found that N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and a monohydroxylated derivative of melatonin were the main products of the reaction between melatonin and ROO. Higher pH or saturation of the medium with molecular oxygen increased the yield of AFMK but did not affect the reaction rate. Melatonin increased the depletion of intracellular GSH mediated by AAPH. Using the HRP/H2O2 as the oxidant system, the addition of melatonin promoted the oxidation of GSH to GSSG. CONCLUSIONS: These results show, for the first time, that melatonin radical is able to oxidize GSH. GENERAL SIGNIFICANCE: We propose that this new property of melatonin could explain or be related to the recently reported pro-oxidant activities of melatonin.
Assuntos
Amidinas/metabolismo , Melatonina/metabolismo , Oxidantes/metabolismo , Peróxidos/metabolismo , Cromatografia Líquida de Alta Pressão , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Espaço Intracelular/metabolismo , Nitrogênio/metabolismo , Oxirredução , Oxigênio/metabolismo , SolubilidadeRESUMO
High levels of oxidative stress were reported in obesity-linked type 2 diabetes and were associated with elevated formation of advanced glycation end products (AGEs). Many studies have focused on the effect of antioxidants on vascular and circulating cells such as macrophages. However, despite the major role of adipocytes in the etiology of diabetes, little is known about the effect of natural antioxidants on adipocyte response to oxidative stress. The present study reports the differential protective effects of plant nutrients toward adipose cells subjected to oxidative stress. Caffeic acid, quercetin, L: -ascorbic acid, and alpha-tocopherol were tested on SW872 liposarcoma cells subjected to a free radical generator or to AGEs. Proliferation, viability, free radical formation, and superoxide dismutase expression were assessed in treated cells. Caffeic acid and quercetin appeared as the most potent antioxidant nutrients. Our findings clearly show a novel antioxidant role for caffeic acid and quercetin at the adipose tissue level. These new data confirm the beneficial role of phytotherapy as an interesting alternative mean for the development of novel therapeutical and nutritional strategy to prevent metabolic disorders inherent to obesity-linked diabetes.