RESUMO
Somatostatin receptors are members of G-protein coupled receptor superfamily. Receptors can be classified into five subtypes, SSTR1 to 5. The highly potent and orally active SSTR2 agonist 7, which had been identified by our group, was found out to have toxicological liabilities such as hERG inhibition and phospholipidosis (PLD). We investigated the relationship between in silico physicochemical properties and hERG and PLD, and explored well-balanced agonists to identify amide 19 and benzimidazole 30. As a result of this exploration, we found out that the value of (cLogP) [2] + (pKa) [2] needs to be less than 110 to mitigate the liabilities.
Assuntos
Amidas/farmacologia , Benzimidazóis/farmacologia , Desenho de Fármacos , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Fosfolipídeos/antagonistas & inibidores , Receptores de Somatostatina/agonistas , Amidas/síntese química , Amidas/química , Benzimidazóis/síntese química , Benzimidazóis/química , Relação Dose-Resposta a Droga , Canais de Potássio Éter-A-Go-Go/metabolismo , Humanos , Estrutura Molecular , Fosfolipídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
During postnatal adaptation pulmonary surfactant may be inactivated by lipopolysaccharide (LPS). We evaluated the effect of surfactant therapy in combination with antibiotic polymyxin B (PxB) in double-hit model of neonatal lung injury. Surfactant (poractant alfa, Curosurf) was exposed to smooth (S) LPS without/with PxB and tested in captive bubble surfactometer. Preterm rabbits received intratracheally saline (control) or S-LPS and were ventilated with 100% oxygen. After 30 min, LPS-treated animals received no treatment, or surfactant (200 mg/kg) without/with 3% PxB; controls received the same dose of surfactant. Animals were ventilated for further 2 h. In vitro, addition of 5% S-LPS to surfactant increased minimum surface tension (γmin) and addition of 1-3% PxB to surfactant/S-LPS mixture restored γmin to low values. Animals only given S-LPS had lower lung compliance and lung gas volume (LGV) compared to surfactant groups. Treatment with surfactant/PxB, but not with surfactant only, restored LGV. Addition of PxB to the surfactant increased the alveolar expansion. S-LPS interferes with surface activity of the pulmonary surfactant and PxB improves the resistance of surfactant to LPS-induced inactivation. In our neonatal model of respiratory distress syndrome surfactant gives positive response even in simultaneous exposure to S-LPS, when enriched with PxB.
Assuntos
Lesão Pulmonar/tratamento farmacológico , Lesão Pulmonar/metabolismo , Polimixina B/farmacologia , Surfactantes Pulmonares/metabolismo , Animais , Animais Recém-Nascidos , Produtos Biológicos/administração & dosagem , Produtos Biológicos/antagonistas & inibidores , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Recém-Nascido , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/fisiopatologia , Complacência Pulmonar/efeitos dos fármacos , Lesão Pulmonar/induzido quimicamente , Masculino , Fosfolipídeos/administração & dosagem , Fosfolipídeos/antagonistas & inibidores , Polimixina B/administração & dosagem , Surfactantes Pulmonares/administração & dosagem , Surfactantes Pulmonares/agonistas , Síndrome do Desconforto Respiratório do Recém-Nascido/tratamento farmacológico , Síndrome do Desconforto Respiratório do Recém-Nascido/metabolismoRESUMO
SUN13837 (1), a fibroblast growth factor receptor modulator, has been an attractive candidate for treating neurodegenerative diseases. However, one of its metabolites, N-benzyl-4-(methylamino)piperidine (BMP), turned out to possess phospholipidosis-inducing potential (PLIP) in vitro. To obtain SUN13837 analogs with reduced phospholipidosis risk, we replaced BMP with other diamines possessing low PLIP. Our effort led to the discovery of compound 6 with increased efficacy. Further structural modifications to reduce hydrogen bond donors afforded 17 with improved brain exposure. Oral administration of 17 at 1 mg/kg once daily for 10 days showed enhanced recovery of coordinated movement in a rat acute stroke model, suggesting that it is a promising follow-up compound for 1 with reduced risk of phospholipidosis.
Assuntos
Diaminas/farmacologia , Fármacos Neuroprotetores/farmacologia , Fosfolipídeos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Animais , Células CACO-2 , Diaminas/síntese química , Diaminas/química , Relação Dose-Resposta a Droga , Humanos , Masculino , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fosfolipídeos/metabolismo , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae), the 'Argentine mistletoe', is a hemiparasite species largely used in folk medicine. The aim of this study was to evaluate the antioxidant activity using inâ vitro, ex vivo, and inâ vivo methods. A screening of phenolics was performed by UV spectroscopy on different fractions. The antioxidant capacity was evaluated inâ vitro by the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) assay on a crude extract (CE), ethyl acetate fraction (EAF), and aqueous fraction (AF). The results suggest that EAF concentrates the antioxidant capacity and was selected for further analysis. Capillary electrophoresis was employed to monitor the individual antioxidant capacity and the potential contributors to this effect. Ex vivo assays showed an efficient inhibition of tert-butyl hydroperoxide-induced rat liver phospholipid oxidation, as well as rat brain autoxidation, and H2 O2 -induced DNA damage in blood monocytes. In vivo, the topical application of EAF significantly decreased skin chemiluminescence in a mice model.
Assuntos
Antioxidantes/farmacologia , Flavonoides/farmacologia , Loranthaceae/química , Fosfolipídeos/antagonistas & inibidores , Extratos Vegetais/farmacologia , Animais , Antioxidantes/química , Antioxidantes/isolamento & purificação , Argentina , Compostos de Bifenilo/antagonistas & inibidores , Dano ao DNA , Feminino , Flavonoides/química , Flavonoides/isolamento & purificação , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Oxirredução , Fosfolipídeos/metabolismo , Picratos/antagonistas & inibidores , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Ratos , Ratos Sprague-Dawley , terc-Butil Hidroperóxido/antagonistas & inibidores , terc-Butil Hidroperóxido/farmacologiaRESUMO
Synchrotron resonance-enhanced infrared atomic force microscopy (RE-AFM-IR) is a near-field photothermal vibrational nanoprobe developed at Diamond Light Source (DLS), capable of measuring mid-infrared absorption spectra with spatial resolution around 100 nm. The present study reports a first application of synchrotron RE-AFM-IR to interrogate biological soft matter at the subcellular level, in this case, on a cellular model of drug-induced phospholipidosis (DIPL). J774A-1 macrophages were exposed to amiodarone (10 µM) or medium for 24 h and chemically fixed. AFM topography maps revealed amiodarone-treated cells with enlarged cytoplasm and very thin regions corresponding to collapsed vesicles. IR maps of the whole cell were analyzed by exploiting the RE-AFM-IR overall signal, i.e., the integrated RE-AFM-IR signal amplitude versus AFM-derived cell thickness, also on lateral resolution around 100 nm. Results show that vibrational band assignment was possible, and all characteristic peaks for lipids, proteins, and DNA/RNA were identified. Both peak ratio and unsupervised chemometric analysis of RE-AFM-IR nanospectra generated from the nuclear and perinuclear regions of untreated and amiodarone-treated cells showed that the perinuclear region (i.e., cytoplasm) of amiodarone-treated cells had significantly elevated band intensities in the regions corresponding to phosphate and carbonyl groups, indicating detection of phospholipid-rich inclusion bodies typical for cells with DIPL. The results of this study are of importance to demonstrate not only the applicability of Synchrotron RE-AFM-IR to soft biological matters with subcellular spatial resolution but also that the spectral information gathered from an individual submicron sample volume enables chemometric identification of treatment and biochemical differences between mammalian cells.
Assuntos
Amiodarona/farmacologia , Antiarrítmicos/farmacologia , Macrófagos/efeitos dos fármacos , Fosfolipídeos/antagonistas & inibidores , Síncrotrons , Temperatura , Animais , Células Cultivadas , Macrófagos/metabolismo , Camundongos , Fosfolipídeos/metabolismo , Processos Fotoquímicos , Espectrofotometria InfravermelhoRESUMO
Within the lipidome oxidized phospholipids (OxPL) form a class of chemically highly reactive metabolites. OxPL are acutely produced in inflamed tissue and act as endogenous, proalgesic (pain-inducing) metabolites. They excite sensory, nociceptive neurons by activating transient receptor potential ion channels, specifically TRPA1 and TRPV1. Under inflammatory conditions, OxPL-mediated receptor potentials even potentiate the action potential firing rate of nociceptors. Targeting OxPL with D-4F, an apolipoprotein A-I mimetic peptide or antibodies like E06, specifically binding oxidized headgroups of phospholipids, can be used to control acute, inflammatory pain syndromes, at least in rodents. With a focus on proalgesic specificities of OxPL, this article discusses, how targeting defined substances of the epilipidome can contribute to mechanism-based therapies against primary and secondary chronic inflammatory or possibly also neuropathic pain.
Assuntos
Analgésicos/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Estresse Oxidativo/fisiologia , Manejo da Dor/métodos , Dor/metabolismo , Fosfolipídeos/metabolismo , Animais , Sistemas de Liberação de Medicamentos/tendências , Humanos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Dor/tratamento farmacológico , Manejo da Dor/tendências , Fosfolipídeos/antagonistas & inibidoresRESUMO
Human phospholipid scramblases are a family of four homologous transmembrane proteins (hPLSCR1-4) mediating phospholipids (PLs) translocation in plasma membrane upon Ca2+ activation. hPLSCR3, the only homologue localized to mitochondria, plays a vital role in mitochondrial structure, function, maintenance, and apoptosis. Upon Ca2+ activation, hPLSCR3 mediates PL translocation at the mitochondrial membrane enhancing t-bid-induced cytochrome c release and apoptosis. Mitochondria are important target organelles for heavy-metals-induced apoptotic signaling cascade and are the central executioner of apoptosis to trigger. Pb2+ and Hg2+ toxicity mediates apoptosis by increased reactive oxygen species (ROS) and cytochrome c release from mitochondria. To discover the role of hPLSCR3 in heavy metal toxicity, hPLSCR3 was overexpressed as a recombinant protein in Escherichia coli Rosetta (DE3) and purified by affinity chromatography. The biochemical assay using synthetic proteoliposomes demonstrated that hPLSCR3 translocated aminophospholipids in the presence of micromolar concentrations of Pb2+ and Hg2+. A point mutation in the Ca2+-binding motif (F258V) led to a â¼60% loss in the functional activity and decreased binding affinities for Pb2+ and Hg2+ implying that the divalent heavy metal ions bind to the Ca2+-binding motif. This was further affirmed by the characteristic spectra observed with stains-all dye. The conformational changes upon heavy metal binding were monitored by circular dichroism, intrinsic tryptophan fluorescence, and light-scattering studies. Our results revealed that Pb2+ and Hg2+ bind to hPLSCR3 with higher affinity than Ca2+ thus mediating scramblase activity. To summarize, this is the first biochemical evidence for heavy metals binding to the mitochondrial membrane protein leading to bidirectional translocation of PLs specifically toward phosphatidylethanolamine.
Assuntos
Apoptose/efeitos dos fármacos , Chumbo/farmacologia , Mercúrio/farmacologia , Mitocôndrias/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/antagonistas & inibidores , Cálcio/química , Cálcio/farmacologia , Relação Dose-Resposta a Droga , Humanos , Chumbo/química , Mercúrio/química , Mitocôndrias/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/isolamento & purificação , Fosfolipídeos/metabolismo , Relação Estrutura-AtividadeRESUMO
Choroidal endothelial cells supply oxygen and nutrients to retinal pigment epithelial (RPE) cells and photoreceptors, recycle metabolites, and dispose of metabolic waste through the choroidal blood circulation. Death of the endothelial cells of the choroid may cause abnormal deposits including unesterified and esterified cholesterol beneath RPE cells and within Bruch's membrane that contribute to the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in older people. Translocator protein (TSPO) is a cholesterol-binding protein that is involved in mitochondrial cholesterol transport and other cellular functions. We have investigated the role of TSPO in choroidal endothelial cells. Immunocytochemistry showed that TSPO was localized to the mitochondria of choroidal endothelial cells. Choroidal endothelial cells exposed to TSPO ligands (Etifoxine or XBD-173) had significantly increased cholesterol efflux, higher expression of cholesterol homeostasis genes (LXRα, CYP27A1, CYP46A1, ABCA1 and ABCG1), and reduced biosynthesis of cholesterol and phospholipids from [14C]acetate, when compared to untreated controls. Treatment with TSPO ligands also resulted in reduced production of reactive oxygen species (ROS), increased antioxidant capacity, and reduced release of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and VEGF) induced by oxidized LDL. These data suggest TSPO ligands may offer promise for the treatment of AMD.
Assuntos
Colesterol/metabolismo , Corioide/efeitos dos fármacos , Lipoproteínas LDL/antagonistas & inibidores , Oxazinas/farmacologia , Purinas/farmacologia , Espécies Reativas de Oxigênio/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Linhagem Celular , Corioide/citologia , Corioide/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Ligantes , Lipoproteínas LDL/farmacologia , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Macaca mulatta , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/biossíntese , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis.
Assuntos
Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Hipercolesterolemia/metabolismo , Inflamação/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/metabolismo , Animais , Estenose da Valva Aórtica/tratamento farmacológico , Estenose da Valva Aórtica/metabolismo , Estenose da Valva Aórtica/patologia , Apoptose , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Colesterol/administração & dosagem , Colesterol/farmacologia , Progressão da Doença , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Hipercolesterolemia/patologia , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Imunoglobulina M/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/patologia , Lipoproteínas LDL/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredução , Fosfolipídeos/química , Fosfolipídeos/imunologia , Fosforilcolina/imunologia , Receptores de LDL/deficiência , Receptores de LDL/genética , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/uso terapêuticoRESUMO
The respiratory system is constantly exposed to pathogens which enter the lungs by inhalation or via blood stream. Lipopolysaccharide (LPS), also named endotoxin, can reach the airspaces as the major component of the outer membrane of Gram-negative bacteria, and lead to local inflammation and systemic toxicity. LPS affects alveolar type II (ATII) cells and pulmonary surfactant and although surfactant molecule has the effective protective mechanisms, excessive amount of LPS interacts with surfactant film and leads to its inactivation. From immunological point of view, surfactant specific proteins (SPs) SP-A and SP-D are best characterized, however, there is increasing evidence on the involvement of SP-B and SP-C and certain phospholipids in immune reactions. In animal models, the instillation of LPS to the respiratory system induces acute lung injury (ALI). It is of clinical importance that endotoxin-induced lung injury can be favorably influenced by intratracheal instillation of exogenous surfactant. The beneficial effect of this treatment was confirmed for both natural porcine and synthetic surfactants. It is believed that the surfactant preparations have anti-inflammatory properties through regulating cytokine production by inflammatory cells. The mechanism by which LPS interferes with ATII cells and surfactant layer, and its consequences are discussed below.
Assuntos
Produtos Biológicos/antagonistas & inibidores , Produtos Biológicos/metabolismo , Lipopolissacarídeos/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/metabolismo , Surfactantes Pulmonares/antagonistas & inibidores , Surfactantes Pulmonares/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Animais , Humanos , Lipopolissacarídeos/toxicidade , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , SuínosRESUMO
The α2 isoform of Na,K-ATPase plays a crucial role in Ca(2+) handling, muscle contraction, and inotropic effects of cardiac glycosides. Thus, structural, functional, and pharmacological comparisons of α1, α2, and α3 are of great interest. In Pichia pastoris membranes expressing human α1ß1, α2ß1, and α3ß1 isoforms, or using the purified isoform proteins, α2 is most easily inactivated by heating and detergent (α2 â« α3 > α1). We have examined an hypothesis that instability of α2 is caused by weak interactions with phosphatidylserine, which stabilizes the protein. Three residues, unique to α2, in trans-membrane segments M8 (Ala-920), M9 (Leu-955), and M10 (Val-981) were replaced by equivalent residues in α1, singly or together. Judged by the sensitivity of the purified proteins to heat, detergent, "affinity" for phosphatidylserine, and stabilization by FXYD1, the triple mutant (A920V/L955F/V981P, called α2VFP) has stability properties close to α1, although single mutants have only modest or insignificant effects. Functional differences between α1 and α2 are unaffected in α2VFP. A compound, 6-pentyl-2-pyrone, isolated from the marine fungus Trichoderma gamsii is a novel probe of specific phospholipid-protein interactions. 6-Pentyl-2-pyrone inactivates the isoforms in the order α2 â« α3 > α1, and α2VFP and FXYD1 protect the isoforms. In native rat heart sarcolemma membranes, which contain α1, α2, and α3 isoforms, a component attributable to α2 is the least stable. The data provide clear evidence for a specific phosphatidylserine binding pocket between M8, M9, and M10 and confirm that the instability of α2 is due to suboptimal interactions with phosphatidylserine. In physiological conditions, the instability of α2 may be important for its cellular regulatory functions.
Assuntos
Isoenzimas/metabolismo , Fosfolipídeos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Estabilidade Enzimática/efeitos dos fármacos , Humanos , Isoenzimas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Mutação , Fosfatidilserinas/antagonistas & inibidores , Fosfatidilserinas/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Pironas/farmacologia , Ratos , Sarcolema/metabolismo , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The antiphospholipid syndrome (APS) is defined by the association of arterial and/or venous thrombosis and/or pregnancy complications with the presence of at least one among the main antiphospholipid antibodies (aPL) (i. e., Lupus anticoagulants, LA, IgG and/or IgM anticardiolipin antibodies, aCL, IgG and/or IgM antiß2-glycoprotein I antibodies, aß2-GPI). Several clinical studies have consistently reported that LA is a stronger risk factor for both arterial and venous thrombosis compared to aCL and aß2-GPI. In particular, LA activity dependent on the first domain of ß2-GPI and triple aPL positivity are associated with the risk of thrombosis and obstetrical complications. Asymptomatic aPL-positive subjects do not require primary thromboprophylaxis. Venous thromboembolism is the most common initial clinical manifestation of APS. To prevent its recurrence indefinite anticoagulation is recommended. Long duration treatment with warfarin or aspirin is used after a first cerebral arterial thrombosis. Low molecular weight heparin (LMWH) with or without aspirin is recommended to reduce the rate of obstetrical complications of APS pregnant women.
Assuntos
Anticoagulantes/administração & dosagem , Síndrome Antifosfolipídica/tratamento farmacológico , Fosfolipídeos/antagonistas & inibidores , Trombose/prevenção & controle , Síndrome Antifosfolipídica/diagnóstico , Feminino , Humanos , Gravidez , Complicações na Gravidez/diagnóstico , Complicações na Gravidez/tratamento farmacológico , Trombose/diagnósticoRESUMO
Diaphanous-related formins (DRFs) are large multi-domain proteins that nucleate and assemble linear actin filaments. Binding of active Rho family proteins to the GTPase-binding domain (GBD) triggers localization at the membrane and the activation of most formins if not all. In recent years GTPase regulation of formins has been extensively studied, but other molecular mechanisms that determine subcellular distribution or regulate formin activity have remained poorly understood. Here, we provide evidence that the activity and localization of mouse formin mDia1 can be regulated through interactions with phospholipids. The phospholipid-binding sites of mDia1 are clusters of positively charged residues in the N-terminal basic domain (BD) and at the C-terminal region. Upon binding to the lipid bilayer the N-terminal region of mDia1 induces strong clustering of phosphatidylinositol-4,5-bisphosphate (PIP(2)) and subsequently inserts into the membrane bilayer thus anchoring mDia1 to the reconstituted plasma membrane. In addition, an interaction of phospholipids with the C-terminal region of mDia1 causes a drastic reduction of its actin filament assembly activity. Our data suggest that the N-terminal phospholipid-binding sites help to anchor formins at the plasma membrane, and the interaction with phospholipids in the C-terminus functions as a switch for transient inactivation.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Proteínas Fetais/metabolismo , Lipídeos de Membrana/fisiologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Fosfolipídeos/fisiologia , Citoesqueleto de Actina/química , Animais , Sítios de Ligação/fisiologia , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/química , Membrana Celular/química , Ativação Enzimática/fisiologia , Proteínas Fetais/antagonistas & inibidores , Proteínas Fetais/química , Forminas , Lipídeos de Membrana/antagonistas & inibidores , Lipídeos de Membrana/química , Camundongos , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/química , Células NIH 3T3 , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/química , Polimerização , Ligação Proteica/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
Regulators of G protein signaling (RGS) proteins are potent negative modulators of G protein signaling and have been proposed as potential targets for small-molecule inhibitor development. We report a high-throughput time-resolved fluorescence resonance energy transfer screen to identify inhibitors of RGS4 and describe the first reversible small-molecule inhibitors of an RGS protein. Two closely related compounds, typified by CCG-63802 [((2E)-2-(1,3-benzothiazol-2-yl)-3-[9-methyl-2-(3-methylphenoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-3-yl]prop-2-enenitrile)], inhibit the interaction between RGS4 and Galpha(o) with an IC(50) value in the low micromolar range. They show selectivity among RGS proteins with a potency order of RGS 4 > 19 = 16 > 8 >> 7. The compounds inhibit the GTPase accelerating protein activity of RGS4, and thermal stability studies demonstrate binding to the RGS but not to Galpha(o). On RGS4, they depend on an interaction with one or more cysteines in a pocket that has previously been identified as an allosteric site for RGS regulation by acidic phospholipids. Unlike previous small-molecule RGS inhibitors identified to date, these compounds retain substantial activity under reducing conditions and are fully reversible on the 10-min time scale. CCG-63802 and related analogs represent a useful step toward the development of chemical tools for the study of RGS physiology.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Transdução de Sinais , Cisteína/antagonistas & inibidores , Cisteína/química , Transferência Ressonante de Energia de Fluorescência , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Fosfolipídeos/antagonistas & inibidores , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/química , Proteínas RGS/metabolismoRESUMO
ErbB4 is a predominant heterodimer for other ErbB receptors in late fetal lung development where it participates in regulating type II cell surfactant synthesis. To further elucidate the role of ErbB4 in pulmonary alveolar epithelial cell function, the authors hypothesized that ErbB4 participates in maintaining adult lung type II cell homeostasis. The authors used small interfering RNA (siRNA) to down-regulate endogenous, ErbB4 receptors in the adult rat lung epithelial L2 cell line and measured neuregulin 1beta (NRG1beta)-, and fibroblast conditioned medium (FCM)-induced effects on L2 cell surfactant phospholipid synthesis and proliferation. Under control conditions, total and phosphorylated ErbB4 were significantly increased after both NRG1beta and FCM treatment, as were surfactant phospholipids synthesis and cell proliferation. Down-regulation of ErbB4 with siRNA reduced stimulation of NRG1beta- and FCM-induced ErbB4 phosphorylation, decreased endogenous surfactant phospholipid synthesis, and blocked NRG1beta- and FCM-stimulated surfactant phospholipid synthesis. NRG1beta- and FCM-induced cell proliferation was not affected. The authors conclude that ErbB4 participates in maintaining adult lung alveolar epithelial cell surfactant synthesis and proliferation with development-specific functions.
Assuntos
Receptores ErbB/metabolismo , Proteínas Associadas a Surfactantes Pulmonares/biossíntese , Mucosa Respiratória/metabolismo , Animais , Contagem de Células , Linhagem Celular , Proliferação de Células , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Receptores ErbB/genética , Feminino , Expressão Gênica/efeitos dos fármacos , Transferência Genética Horizontal , Masculino , Neuregulina-1/genética , Neuregulina-1/metabolismo , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/biossíntese , Fosforilação , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor ErbB-4 , Mucosa Respiratória/citologia , Mucosa Respiratória/efeitos dos fármacosRESUMO
Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.
Assuntos
Lipídeos/antagonistas & inibidores , Proteína A Associada a Surfactante Pulmonar/farmacologia , Surfactantes Pulmonares/antagonistas & inibidores , Surfactantes Pulmonares/metabolismo , Soroalbumina Bovina/farmacologia , Adsorção , Ar , Animais , Bovinos , Humanos , Microscopia de Força Atômica , Microscopia de Fluorescência , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/metabolismo , Pressão , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Soroalbumina Bovina/uso terapêutico , Propriedades de Superfície , Água/químicaRESUMO
Histone deacetylases (HDACs) are able to control also the acetylation of tubulin. In the present experiments the effect of trichostatin A (TSA), a HDAC inhibitor was studied on the incorporation of 3H-palmitic acid and 32P to the phospholipids (PI, PIP, PS, PC, PA, PE) of Tetrahymena pyriformis, considering earlier observations on the microtubular system's influence on signalling in this unicellular eukaryote. Treatment with 1, 5, or 10 microM TSA was studied. The incorporation of hydrophobic tail component, palmitic acid was inhibited in a concentration dependent manner into all the phospholipids, except for PA, where the incorporation was increased. 32P incorporation was also inhibited. The possible relation between the microtubular system and signalling is discussed.
Assuntos
Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Ácido Palmítico/metabolismo , Fosfatos/metabolismo , Fosfolipídeos/biossíntese , Tetrahymena pyriformis/enzimologia , Animais , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Ácido Palmítico/antagonistas & inibidores , Fosfolipídeos/antagonistas & inibidores , Radioisótopos de Fósforo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Tetrahymena pyriformis/efeitos dos fármacos , Trítio/metabolismoRESUMO
Phospholipase A(2)s (PLA(2)s) are a superfamily of enzymes involved in production of a wide variety of lipid mediators, including arachidonic acid, lysophospholipids, platelet activation factor, and eicosanoids. Fluorescence-based, real-time cellular activity assays for PLA(2)s have been developed as a tool for studying the function and spatiotemporal regulation of PLA(2)s. Recent progress in fluorogenic phospholipid design, genetic methods, and multiphoton multichannel microscopy allows simultaneous and continuous measurement of cellular localization and activity of PLA(2) in an isoform-selective manner. These assays should aid in elucidating the physiological roles and regulatory mechanisms of diverse PLA(2) isoforms, developing isoform-specific PLA(2) inhibitors, and analyzing lipidomics data of PLA(2) products.
Assuntos
Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Animais , Membrana Celular/enzimologia , Inibidores Enzimáticos/farmacologia , Fluoretos , Humanos , Inflamação/enzimologia , Inflamação/fisiopatologia , Insulina/fisiologia , Cinética , Masculino , Fosfatidilcolinas , Inibidores de Fosfolipase A2 , Fosfolipídeos/antagonistas & inibidores , Fosfolipídeos/síntese química , Transdução de Sinais , Espermatogênese , Especificidade por SubstratoRESUMO
Activated protein C (APC) anticoagulant activity and the ability to be inhibited by auto-antibodies associated with thrombosis are strongly augmented by the presence of phosphatidylethanolamine (PE) and phospholipid oxidation. beta(2)-glycoprotein I (beta(2)-GPI) is a major antigen for antiphospholipid antibodies present in patients with the antiphospholipid syndrome. We therefore investigated whether anti-beta(2)-GPI monoclonal antibodies (mAbs) could inhibit APC with similar membrane specificity. Five mouse mAbs that reacted with different epitopes on beta(2)-GPI were examined. Each inhibited the PE-, phospholipid oxidation-dependent enhancement of APC anticoagulant activity and required antibody divalency. A chimeric APC that retains anticoagulant activity but is relatively unaffected by protein S, PE, or oxidation was not inhibited by the antibodies. In purified systems, anti-beta(2)-GPI mAb inhibition of factor Va inactivation was greater in the presence of protein S and required beta(2)-GPI. Surprisingly, although the mAbs did increase beta(2)-GPI affinity for membranes, PE and oxidation had little influence on the affinity of the beta(2)-GPI antibody complex for the membrane vesicles. We conclude that antibodies to beta(2)-GPI inhibit APC function specifically and contribute to a hypercoaguable state by disrupting specific protein-protein interactions induced by oxidation of PE-containing membranes.
Assuntos
Anticorpos Antifosfolipídeos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticoagulantes/antagonistas & inibidores , Glicoproteínas/imunologia , Fosfolipídeos/metabolismo , Proteína C/efeitos dos fármacos , Animais , Anticoagulantes/metabolismo , Relação Dose-Resposta a Droga , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Oxirredução , Fosfolipídeos/antagonistas & inibidores , Proteína C/metabolismo , Fatores de Tempo , beta 2-Glicoproteína IRESUMO
Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.