RESUMO
PURPOSE: High-grade gliomas (HGG) carry a poor prognosis, with glioblastoma accounting for almost 50% of primary brain malignancies in the elderly. Unfortunately, despite the use of multiple treatment modalities, the prognosis remains poor in this population. Our preclinical studies suggest that the presence of aromatase expression, encoded by CYP19A1, is significantly upregulated in HGGs. Remarkably, we find that letrozole (LTZ), an FDA-approved aromatase inhibitor, has marked activity against HGGs. PATIENTS AND METHODS: We conducted a phase 0/I single-center clinical trial (NCT03122197) to assess the tumoral availability, pharmacokinetics (PK), safety, and tolerability of LTZ in recurrent patients with HGG. Planned dose cohorts included 2.5, 5, 10, 12.5, 15, 17.5, and 20 mg of LTZ administered daily pre- and postsurgery or biopsy. Tumor samples were assayed for LTZ content and relevant biomarkers. The recommended phase 2 dose (R2PD) was determined as the dose that resulted in predicted steady-state tumoral extracellular fluid (ECF; Css,ecf) >2 µmol/L and did not result in ≥33% dose-limiting adverse events (AE) assessed using CTCAE v5.0. RESULTS: Twenty-one patients were enrolled. Common LTZ-related AEs included fatigue, nausea, musculoskeletal, anxiety, and dysphoric mood. No DLTs were observed. The 15 mg dose achieved a Css,ecf of 3.6 ± 0.59 µmol/L. LTZ caused dose-dependent inhibition of estradiol synthesis and modulated DNA damage pathways in tumor tissues as evident using RNA-sequencing analysis. CONCLUSIONS: On the basis of safety, brain tumoral PK, and mechanistic data, 15 mg daily is identified as the RP2D for future trials.
Assuntos
Neoplasias Encefálicas , Glioma , Letrozol , Gradação de Tumores , Recidiva Local de Neoplasia , Humanos , Letrozol/administração & dosagem , Letrozol/farmacocinética , Letrozol/uso terapêutico , Letrozol/efeitos adversos , Feminino , Glioma/tratamento farmacológico , Glioma/patologia , Pessoa de Meia-Idade , Masculino , Idoso , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/patologia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinéticaRESUMO
PURPOSE: Emerging evidence suggests that 5' Adenosine Monophosphate-Activated Protein Kinase (AMPK), a key regulator of cellular bioenergetics, is a novel target for the treatment of glioblastoma (GBM), a lethal brain tumor. SBI-0206965, an aminopyrimidine derivative, is a potent AMPK inhibitor being investigated for the treatment of GBM. Here we characterized the systemic and brain pharmacokinetics (PK) and hepatic metabolism of SBI-0206965. METHODS: We performed intracerebral microdialysis to determine brain partitioning of SBI-0206965 in jugular vein cannulated rats. We assessed systemic PK of SBI-0206965 in rats and C57BL/6 mice following oral administration. Employing human, mouse, and rat liver microsomes we characterized the metabolism of SBI-0206965. RESULTS: SBI-0206965 is quickly absorbed, achieving plasma and brain extracellular fluid (ECF) peak levels within 0.25 - 0.65 h. Based on the ratio of Cmax and AUC in brain ECF to plasma (corrected for protein binding), brain partitioning is ~ 0.6-0.9 in rats. However, the compound has a short elimination half-life (1-2 h) and low relative oral bioavailability (~ 0.15). The estimated in-vitro hepatic intrinsic clearance of SBI-0206965 in mouse, rat and human was 325, 76 and 68 mL/min/kg, respectively. SBI-0206965 metabolites included desmethylated products, and the metabolism was strongly inhibited by ketoconazole, a CYP3A inhibitor. CONCLUSION: SBI-0206965 has adequate brain permeability but low relative oral bioavailability which may be due to rapid hepatic metabolism, likely catalyzed by CYP3A enzymes. Our observations will facilitate further development of SBI-0206965, and/or other structurally related molecules, for the treatment of GBM and other brain tumors.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Benzamidas , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Drogas em Investigação , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pirimidinas , RatosRESUMO
Under aerobic conditions the production of Reactive Oxygen Species (ROS) by electron transport chains is unavoidable, and occurs in both autotrophic and heterotrophic organisms. In photosynthetic organisms both Photosystem II (PS II) and Photosystem I (PS I), in addition to the cytochrome b6/f complex, are demonstrated sources of ROS. All of these membrane protein complexes exhibit oxidative damage when isolated from field-grown plant material. An additional possible source of ROS in PS I and PS II is the distal, chlorophyll-containing light-harvesting array LHC II, which is present in both photosystems. These serve as possible sources of 1O2 produced by the interaction of 3O2 with 3chl* produced by intersystem crossing. We have hypothesized that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in a subset of the spinach LHC II proteins (Lhcb1 and Lhcb2) that were associated with either PS II membranes (i.e. BBYs) or PS I-LHC I-LHC II membranes, both of which were isolated from field-grown spinach. We identified oxidatively modified residues by high-resolution tandem mass spectrometry. Interestingly, two different patterns of oxidative modification were evident for the Lhcb1 and Lhcb2 proteins from these different sources. In the LHC II associated with PS II membranes, oxidized residues were identified to be located on the stromal surface of Lhcb1 and, to a much lesser extent, Lhcb2. Relatively few oxidized residues were identified as buried in the hydrophobic core of these proteins. The LHC II associated with PS I-LHC I-LHC II membranes, however, exhibited fewer surface-oxidized residues but, rather a large number of oxidative modifications buried in the hydrophobic core regions of both Lhcb1 and Lhcb2, adjacent to the chlorophyll prosthetic groups. These results appear to indicate that ROS, specifically 1O2, can modify the Lhcb proteins associated with both photosystems and that the LHC II associated with PS II membranes represent a different population from the LHC II associated with PS I-LHC I-LHC II membranes.
Assuntos
Complexos de Proteínas Captadores de Luz , Complexo de Proteína do Fotossistema II , Aminoácidos/metabolismo , Clorofila/metabolismo , Complexo Citocromos b6f/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Estresse Oxidativo , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)-mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias , Estearoil-CoA Dessaturase , Animais , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metabolismo dos Lipídeos , Lipogênese , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
Photosystem II (PSII) is an intrinsic membrane protein complex that functions as a light-driven water:plastoquinone oxidoreductase in oxygenic photosynthesis. Electron transport in PSII is associated with formation of reactive oxygen species (ROS) responsible for oxidative modifications of PSII proteins. In this study, oxidative modifications of the D1 and D2 proteins by the superoxide anion (O2â¢-) and the hydroxyl (HOâ¢) radicals were studied in WT and a tocopherol cyclase (vte1) mutant, which is deficient in the lipid-soluble antioxidant α-tocopherol. In the absence of this antioxidant, high-resolution tandem mass spectrometry was used to identify oxidation of D1:130E to hydroxyglutamic acid by O2â¢- at the PheoD1 site. Additionally, D1:246Y was modified to either tyrosine hydroperoxide or dihydroxyphenylalanine by O2â¢- and HOâ¢, respectively, in the vicinity of the nonheme iron. We propose that α-tocopherol is localized near PheoD1 and the nonheme iron, with its chromanol head exposed to the lipid-water interface. This helps to prevent oxidative modification of the amino acid's hydrogen that is bonded to PheoD1 and the nonheme iron (via bicarbonate), and thus protects electron transport in PSII from ROS damage.
Assuntos
Aminoácidos/química , Arabidopsis/enzimologia , Complexo de Proteína do Fotossistema II/química , Superóxidos/química , Tilacoides/enzimologia , alfa-Tocoferol/química , Aminoácidos/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Sítios de Ligação , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Transferases Intramoleculares/química , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Ferro/química , Ferro/metabolismo , Luz , Modelos Moleculares , Mutação , Oxirredução , Oxigênio/química , Oxigênio/metabolismo , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Superóxidos/metabolismo , Termodinâmica , Thermosynechococcus/enzimologia , Thermosynechococcus/genética , Thermosynechococcus/efeitos da radiação , Tilacoides/genética , Tilacoides/efeitos da radiação , alfa-Tocoferol/metabolismoRESUMO
Reactive oxygen species (ROS) production is an unavoidable byproduct of electron transport under aerobic conditions. Photosystem II (PS II), the cytochrome b6/f complex and Photosystem I (PS I) are all demonstrated sources of ROS. It has been proposed that PS I produces substantial levels of a variety of ROS including O2.-, 1O2, H2O2 and, possibly, â¢OH; however, the site(s) of ROS production within PS I has been the subject of significant debate. We hypothesize that amino acid residues close to the sites of ROS generation will be more susceptible to oxidative modification than distant residues. In this study, we have identified oxidized amino acid residues in spinach PS I which was isolated from field-grown spinach. The modified residues were identified by high-resolution tandem mass spectrometry. As expected, many of the modified residues lie on the surface of the complex. However, a well-defined group of oxidized residues, both buried and surface-exposed, lead from the chl a' of P700 to the surface of PS I. These residues (PsaB: 609F, 611E, 617M, 619W, 620L, and PsaF: 139L, 142A,143D) may identify a preferred route for ROS, probably 1O2, to egress the complex from the vicinity of P700. Additionally, two buried residues located in close proximity to A1B (PsaB:712H and 714S) were modified, which appears consistent with A1B being a source of O2.-. Surprisingly, no oxidatively modified residues were identified in close proximity to the 4Fe-FS clusters FX, FA or FB. These cofactors had been identified as principal targets for ROS damage in the photosystem. Finally, a large number of residues located in the hydrophobic cores of Lhca1-Lhca4 are oxidatively modified. These appear to be the result of 1O2 production by the distal antennae for the photosystem.
Assuntos
Aminoácidos/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Spinacia oleracea/metabolismo , Sequência de Aminoácidos , Complexos de Proteínas Captadores de Luz/química , Modelos Moleculares , Oxirredução , Complexo de Proteína do Fotossistema I/químicaRESUMO
BACKGROUND: Several tyrosine kinase inhibitors (TKIs) developed as anti-cancer drugs, also have anti-viral activity due to their ability to disrupt productive replication and dissemination in infected cells. Consequently, such drugs are attractive candidates for "repurposing" as anti-viral agents. However, clinical evaluation of therapeutics against infectious agents associated with high mortality, but low or infrequent incidence, is often unfeasible. The United States Food and Drug Administration formulated the "Animal Rule" to facilitate use of validated animal models for conducting anti-viral efficacy studies. METHODS: To enable such efficacy studies of two clinically approved TKIs, nilotinib, and imatinib, we first conducted comprehensive pharmacokinetic (PK) studies in relevant rodent and non-rodent animal models. PK of these agents following intravenous and oral dosing were evaluated in C57BL/6 mice, prairie dogs, guinea pigs and Cynomolgus monkeys. Plasma samples were analyzed using an LC-MS/MS method. Secondarily, we evaluated the utility of allometry-based inter-species scaling derived from previously published data to predict the PK parameters, systemic clearance (CL) and the steady state volume of distribution (Vss) of these two drugs in prairie dogs, an animal model not tested thus far. RESULTS: Marked inter-species variability in PK parameters and resulting oral bioavailability was observed. In general, elimination half-lives of these agents in mice and guinea pigs were much shorter (1-3 h) relative to those in larger species such as prairie dogs and monkeys. The longer nilotinib elimination half-life in prairie dogs (i.v., 6.5 h and oral, 7.5 h), facilitated multiple dosing PK and safety assessment. The allometry-based predicted values of the Vss and CL were within 2.0 and 2.5-fold, respectively, of the observed values. CONCLUSIONS: Our results suggest that prairie dogs and monkeys may be suitable rodent and non-rodent species to perform further efficacy testing of these TKIs against orthopoxvirus infections. The use of rodent models such as C57BL/6 mice and guinea pigs for assessing pre-clinical anti-viral efficacy of these two TKIs may be limited due to short elimination and/or low oral bioavailability. Allometry-based correlations, derived from existing literature data, may provide initial estimates, which may serve as a useful guide for pre-clinical PK studies in untested animal models.
Assuntos
Antineoplásicos/farmacocinética , Antivirais/farmacocinética , Mesilato de Imatinib/farmacocinética , Proteínas Tirosina Quinases/farmacocinética , Pirimidinas/farmacocinética , Administração Intravenosa , Administração Oral , Animais , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Cobaias , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , SciuridaeRESUMO
The cytochrome b 6 f complex of oxygenic photosynthesis produces substantial levels of reactive oxygen species (ROS). It has been observed that the ROS production rate by b 6 f is 10-20 fold higher than that observed for the analogous respiratory cytochrome bc1 complex. The types of ROS produced (O2â¢-, 1O2, and, possibly, H2O2) and the site(s) of ROS production within the b 6 f complex have been the subject of some debate. Proposed sources of ROS have included the heme b p , PQ pâ¢- (possible sources for O2â¢-), the Rieske iron-sulfur cluster (possible source of O2â¢- and/or 1O2), Chl a (possible source of 1O2), and heme c n (possible source of O2â¢- and/or H2O2). Our working hypothesis is that amino acid residues proximal to the ROS production sites will be more susceptible to oxidative modification than distant residues. In the current study, we have identified natively oxidized amino acid residues in the subunits of the spinach cytochrome b 6 f complex. The oxidized residues were identified by tandem mass spectrometry using the MassMatrix Program. Our results indicate that numerous residues, principally localized near p-side cofactors and Chl a, were oxidatively modified. We hypothesize that these sites are sources for ROS generation in the spinach cytochrome b 6 f complex.
Assuntos
Aminoácidos/metabolismo , Complexo Citocromos b6f/química , Complexo Citocromos b6f/metabolismo , Spinacia oleracea/metabolismo , Aminoácidos/química , Sítios de Ligação , Heme/química , Heme/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismoRESUMO
The Photosystem II reaction center is vulnerable to photoinhibition. The D1 and D2 proteins, lying at the core of the photosystem, are susceptible to oxidative modification by reactive oxygen species that are formed by the photosystem during illumination. Using spin probes and EPR spectroscopy, we have determined that both O2â¢- and HO⢠are involved in the photoinhibitory process. Using tandem mass spectroscopy, we have identified a number of oxidatively modified D1 and D2 residues. Our analysis indicates that these oxidative modifications are associated with formation of HO⢠at both the Mn4O5Ca cluster and the nonheme iron. Additionally, O2â¢- appears to be formed by the reduction of O2 at either PheoD1 or QA Early oxidation of D1:332H, which is coordinated with the Mn1 of the Mn4O5Ca cluster, appears to initiate a cascade of oxidative events that lead to the oxidative modification of numerous residues in the C termini of the D1 and D2 proteins on the donor side of the photosystem. Oxidation of D2:244Y, which is a bicarbonate ligand for the nonheme iron, induces the propagation of oxidative reactions in residues of the D-de loop of the D2 protein on the electron acceptor side of the photosystem. Finally, D1:130E and D2:246M are oxidatively modified by O2â¢- formed by the reduction of O2 either by PheoD1â¢- or QAâ¢- The identification of specific amino acid residues oxidized by reactive oxygen species provides insights into the mechanism of damage to the D1 and D2 proteins under light stress.
Assuntos
Aminoácidos/metabolismo , Oxirredução , Complexo de Proteína do Fotossistema II/metabolismo , Aminoácidos/química , Antioxidantes/metabolismo , Cloretos/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radical Hidroxila/metabolismo , Espectrometria de Massas , Modelos Moleculares , Conformação Molecular , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/química , Espécies Reativas de Oxigênio/metabolismoRESUMO
We have used protein cross-linking with the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and radiolytic footprinting coupled with high-resolution tandem mass spectrometry, to examine the structure of higher-plant PsbO when it is bound to Photosystem II. Twenty intramolecular cross-linked residue pairs were identified. On the basis of this cross-linking data, spinach PsbO was modeled using the Thermosynechococcus vulcanus PsbO structure as a template, with the cross-linking distance constraints incorporated using the MODELLER program. Our model of higher-plant PsbO identifies several differences between the spinach and cyanobacterial proteins. The N-terminal region is particularly interesting, as this region has been suggested to be important for oxygen evolution and for the specific binding of PsbO to Photosystem II. Additionally, using radiolytic mapping, we have identified regions on spinach PsbO that are shielded from the bulk solvent. These domains may represent regions on PsbO that interact with other components, as yet unidentified, of the photosystem.
Assuntos
Reagentes de Ligações Cruzadas , Cianobactérias/metabolismo , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Radiólise de Impulso , Spinacia oleracea/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Cianobactérias/crescimento & desenvolvimento , Espectrometria de Massas , Modelos Moleculares , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Conformação Proteica , Pegadas de Proteínas , Homologia de Sequência de Aminoácidos , Spinacia oleracea/crescimento & desenvolvimento , SíncrotronsRESUMO
UNLABELLED: Recent estimates suggest that >300 million people are afflicted by serious fungal infections worldwide. Current antifungal drugs are static and toxic and/or have a narrow spectrum of activity. Thus, there is an urgent need for the development of new antifungal drugs. The fungal sphingolipid glucosylceramide (GlcCer) is critical in promoting virulence of a variety of human-pathogenic fungi. In this study, we screened a synthetic drug library for compounds that target the synthesis of fungal, but not mammalian, GlcCer and found two compounds [N'-(3-bromo-4-hydroxybenzylidene)-2-methylbenzohydrazide (BHBM) and its derivative, 3-bromo-N'-(3-bromo-4-hydroxybenzylidene) benzohydrazide (D0)] that were highly effective in vitro and in vivo against several pathogenic fungi. BHBM and D0 were well tolerated in animals and are highly synergistic or additive to current antifungals. BHBM and D0 significantly affected fungal cell morphology and resulted in the accumulation of intracellular vesicles. Deep-sequencing analysis of drug-resistant mutants revealed that four protein products, encoded by genes APL5, COS111, MKK1, and STE2, which are involved in vesicular transport and cell cycle progression, are targeted by BHBM. IMPORTANCE: Fungal infections are a significant cause of morbidity and mortality worldwide. Current antifungal drugs suffer from various drawbacks, including toxicity, drug resistance, and narrow spectrum of activity. In this study, we have demonstrated that pharmaceutical inhibition of fungal glucosylceramide presents a new opportunity to treat cryptococcosis and various other fungal infections. In addition to being effective against pathogenic fungi, the compounds discovered in this study were well tolerated by animals and additive to current antifungals. These findings suggest that these drugs might pave the way for the development of a new class of antifungals.
Assuntos
Antifúngicos/isolamento & purificação , Antifúngicos/farmacologia , Compostos de Benzil/isolamento & purificação , Compostos de Benzil/farmacologia , Vias Biossintéticas/efeitos dos fármacos , Fungos/efeitos dos fármacos , Esfingolipídeos/biossíntese , Animais , Antifúngicos/efeitos adversos , Antifúngicos/toxicidade , Compostos de Benzil/efeitos adversos , Compostos de Benzil/toxicidade , Candidíase/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Contagem de Colônia Microbiana , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos/métodos , Sinergismo Farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Fungos/citologia , Fungos/metabolismo , Fungos/fisiologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Esfingolipídeos/antagonistas & inibidores , Resultado do TratamentoRESUMO
Betahistine, a potent histamine H3 receptor antagonist, is being developed for the treatment of attention deficit hyperactivity disorder (ADHD) that manifests with symptoms such as hyperactivity, impulsivity and inattention. This study describes the pharmacokinetics of betahistine in ADHD subjects at doses higher than 50 mg. These assessments were made during a randomized, placebo-controlled, single blind, dose escalation study to determine the safety, tolerability and pharmacokinetics of once daily doses of 50 mg, 100 mg and 200 mg of betahistine in subjects with ADHD. Plasma levels of 2-pyridylacetic acid (2-PAA), a major metabolite of betahistine were quantified using a validated LC-MS/MS method and used for pharmacokinetic analysis and dose proportionality of betahistine. A linear relationship was observed in Cmax and AUC0-4 of 2-PAA with the betahistine dose (R2 0.9989 and 0.9978, respectively) and dose proportionality coefficients (ß) for the power model were 0.8684 (Cmax) and 1.007 (AUC0-4). A population pharmacokinetic model with first-order absorption of betahistine and metabolism to 2-PAA, followed by a first-order elimination of 2-PAA provides estimates of clearance that underscored the linear increase in systemic exposure with dose. There were no serious adverse events reported in the study, betahistine was safe and well tolerated at all the dose levels tested.
Assuntos
Acetatos/administração & dosagem , Acetatos/farmacocinética , Transtorno do Deficit de Atenção com Hiperatividade/sangue , Transtorno do Deficit de Atenção com Hiperatividade/tratamento farmacológico , beta-Histina/administração & dosagem , beta-Histina/farmacocinética , Piridinas/administração & dosagem , Piridinas/farmacocinética , Acetatos/efeitos adversos , Administração Oral , Adulto , beta-Histina/efeitos adversos , Tontura/induzido quimicamente , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agitação Psicomotora/etiologia , Piridinas/efeitos adversos , Método Simples-Cego , Adulto JovemRESUMO
Protein cross-linking and radiolytic footprinting coupled with high-resolution mass spectrometry were used to examine the structure of PsbP and PsbQ when they are bound to Photosystem II. In its bound state, the N-terminal 15-amino-acid residue domain of PsbP, which is unresolved in current crystal structures, interacts with domains in the C terminus of the protein. These interactions may serve to stabilize the structure of the N terminus and may facilitate PsbP binding and function. These interactions place strong structural constraints on the organization of PsbP when associated with the Photosystem II complex. Additionally, amino acid residues in the structurally unresolved loop 3A domain of PsbP ((90)K-(107)V), (93)Y and (96)K, are in close proximity (≤ 11.4 Å) to the N-terminal (1)E residue of PsbQ. These findings are the first, to our knowledge, to identify a putative region of interaction between these two components. Cross-linked domains within PsbQ were also identified, indicating that two PsbQ molecules can interact in higher plants in a manner similar to that observed by Liu et al. [(2014) Proc Natl Acad Sci 111(12):4638-4643] in cyanobacterial Photosystem II. This interaction is consistent with either intra-Photosystem II dimer or inter-Photosystem II dimer models in higher plants. Finally, OH(â¢) produced by synchrotron radiolysis of water was used to oxidatively modify surface residues on PsbP and PsbQ. Domains on the surface of both protein subunits were resistant to modification, indicating that they were shielded from water and appear to define buried regions that are in contact with other Photosystem II components.
Assuntos
Complexo de Proteína do Fotossistema II/química , Spinacia oleracea/enzimologia , Reagentes de Ligações Cruzadas , Cristalografia por Raios X , Hidróxidos/química , Complexo de Proteína do Fotossistema II/metabolismo , Pegadas de Proteínas/métodos , Estrutura Quaternária de Proteína , Estrutura Terciária de ProteínaRESUMO
The cariogenic bacterium Streptococcus mutans is an important dental pathogen that forms biofilms on tooth surfaces, which provide a protective niche for the bacterium where it secretes organic acids leading to the demineralization of tooth enamel. Lipids, especially glycolipids are likely to be key components of these biofilm matrices. The UA159 strain of S. mutans was among the earliest microorganisms to have its genome sequenced. While the lipids of other S. mutans strains have been identified and characterized, lipid analyses of UA159 have been limited to a few studies on its fatty acids. Here we report the structures of the four major glycolipids from stationary-phase S. mutans UA159 cells grown in standing cultures. These were shown to be monoglucosyldiacylglycerol (MGDAG), diglucosyldiacylglycerol (DGDAG), diglucosylmonoacylglycerol (DGMAG) and, glycerophosphoryldiglucosyldiacylglycerol (GPDGDAG). The structures were determined by high performance thin-layer chromatography, mass spectrometry and nuclear magnetic resonance spectroscopy. The glycolipids were identified by accurate, high resolution, and tandem mass spectrometry. The identities of the sugar units in the glycolipids were determined by a novel and highly efficient NMR method. All sugars were shown to have alpha-glycosidic linkages and DGMAG was shown to be acylated in the sn-1 position by NMR. This is the first observation of unsubstituted DGMAG in any organism and the first mass spectrometry data for GPDGDAG.
Assuntos
Glicolipídeos/química , Streptococcus mutans/química , Cromatografia em Camada Fina , Placa Dentária/microbiologia , Glicolipídeos/isolamento & purificação , Glicosilação , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Monoglicerídeos/química , Monoglicerídeos/isolamento & purificaçãoRESUMO
Photosystem II uses water as an enzymatic substrate. It has been hypothesized that this water is vectored to the active site for water oxidation via water channels that lead from the surface of the protein complex to the Mn4O5Ca metal cluster. The radiolysis of water by synchrotron radiation produces amino acid residue-modifying OH(â¢) and is a powerful technique to identify regions of proteins that are in contact with water. In this study, we have used this technique to oxidatively modify buried amino acid residues in higher plant Photosystem II membranes. Fourier transform ion cyclotron resonance mass spectrometry was then used to identify these oxidized amino acid residues that were located in several core Photosystem II subunits (D1, D2, CP43, and CP47). While, as expected, the majority of the identified oxidized residues (≈75%) are located on the solvent-exposed surface of the complex, a number of buried residues on these proteins were also modified. These residues form groups which appear to lead from the surface of the complex to the Mn4O5Ca cluster. These residues may be in contact with putative water channels in the photosystem. These results are discussed within the context of a number of largely computational studies that have identified putative water channels in Photosystem II.
Assuntos
Complexo de Proteína do Fotossistema II/química , Spinacia oleracea/enzimologia , Água/química , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodosRESUMO
The opportunistic pathogen Pseudomomas aeruginosa produces multiple pigments during in vitro culture and in vivo during colonization of burn wounds and in the airways of cystic fibrosis (CF) patients. One pigment is a deep 'merlot'-coloured compound known as aeruginosin A (AA). However, the red pigment(s) of P. aeruginosa are often collectively called pyorubrin, of which there is no known chemical composition. Here, we purified and confirmed by MS and assessed the physicochemical properties of AA (2-amino-6-carboxy-10-methylphenazinium betaine) by first focusing on its ability to redox-cycle using cyclic voltammetry and its spectroscopic (as well as fluorescent) properties, experiments that were conducted at physiological pH. AA exhibited reversible electrochemistry at a glassy carbon electrode within a potential range of -500 to -200 mV. Electrochemical anodic and cathodic peak currents were observed at -327 and -360 mV, respectively, with a low formal reduction potential of -343.5 mV versus Ag/AgCl. AA absorbed at 516 nm and fluoresced at 606 nm. Results from the spectro-electrochemistry of pyorubrin revealed that its strongest fluorescence was in its parent or oxidized form. Production of AA by P. aeruginosa was found to be controlled by the rhl component of the intercellular signalling system known as quorum sensing and was produced maximally during the stationary growth phase. However, unlike its downstream blue redox-active toxin, pyocyanin, AA had no adverse effects on methicillin-resistant Staphylococcus aureus USA300, Escherichia coli DH5-α or human keratinocytes. We close with some thoughts on the potential commercial use(s) of AA.
Assuntos
Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Pigmentos Biológicos/química , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/fisiologia , Células Cultivadas , Eletroquímica , Escherichia coli/efeitos dos fármacos , Fluorescência , Humanos , Queratinócitos/efeitos dos fármacos , Espectrometria de Massas , Compostos Orgânicos/isolamento & purificação , Oxirredução , Pigmentos Biológicos/isolamento & purificação , Percepção de Quorum , Staphylococcus aureus/efeitos dos fármacosRESUMO
Under a variety of stress conditions, Photosystem II produces reactive oxygen species on both the reducing and oxidizing sides of the photosystem. A number of different sites including the Mn4O5Ca cluster, P680, PheoD1, QA, QB and cytochrome b559 have been hypothesized to produce reactive oxygen species in the photosystem. In this communication using Fourier-transform ion cyclotron resonance mass spectrometry we have identified several residues on the D1 and D2 proteins from spinach which are oxidatively modified and in close proximity to QA (D1 residues (239)F, (241)Q, (242)E and the D2 residues (238)P, (239)T, (242)E and (247)M) and PheoD1 (D1 residues (130)E, (133)L and (135)F). These residues may be associated with reactive oxygen species exit pathways located on the reducing side of the photosystem, and their modification may indicate that both QA and PheoD1 are sources of reactive oxygen species on the reducing side of Photosystem II.
Assuntos
Aminoácidos , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Oxirredução , Spinacia oleracea/enzimologia , Spinacia oleracea/crescimento & desenvolvimento , Spinacia oleracea/metabolismoRESUMO
As a light-driven water-plastoquinone oxidoreductase, Photosystem II produces molecular oxygen as an enzymatic product. Additionally, under a variety of stress conditions, reactive oxygen species are produced at or near the active site for oxygen evolution. In this study, Fourier-transform ion cyclotron resonance mass spectrometry was used to identify oxidized amino acid residues located in several core Photosystem II proteins (D1, D2, CP43, and CP47) isolated from spinach Photosystem II membranes. While the majority of these oxidized residues (81%) are located on the oxygenated solvent-exposed surface of the complex, several residues on the CP43 protein ((354)E, (355)T, (356)M, and (357)R) which are in close proximity (<15 Å) to the Mn(4)CaO(5) active site are also modified. These residues appear to be associated with putative oxygen/reactive oxygen species exit channel(s) in the photosystem. These results are discussed within the context of a number of computational studies which have identified putative oxygen channels within the photosystem.
Assuntos
Aminoácidos/química , Cálcio/química , Manganês/química , Oxigênio/química , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Domínio Catalítico , Análise de Fourier , Espectrometria de Massas , Modelos Moleculares , Oxirredução , Espécies Reativas de Oxigênio/química , Spinacia oleraceaRESUMO
Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of â¼7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.