RESUMO
INTRODUCTION: Water homoeostasis is achieved by secretion of the peptide hormones arginine vasopressin (AVP) and oxytocin (OXT) that are synthesized by separate populations of magnocellular neurones (MCNs) in the supraoptic and paraventricular (PVN) nuclei of the hypothalamus. To further understand the molecular mechanisms that facilitate biosynthesis of AVP and OXT by MCNs, we have explored the spatiotemporal dynamic, both mRNA and protein expression, of two genes identified by our group as being important components of the osmotic defence response: Caprin2 and Creb3l1. METHODS: By RNA in situ hybridization and immunohistochemistry, we have characterized the expression of Caprin2 and Creb3l1 in MCNs in the basal state, in response to dehydration, and during rehydration in the rat. RESULTS: We found that Caprin2 and Creb3l1 are expressed in AVP and OXT MCNs and in response to dehydration expression increases in both MCN populations. Protein levels mirror the increase in transcript levels for both CREB3L1 and CAPRIN2. In view of increased CREB3L1 and CAPRIN2 expression in OXT neurones by dehydration, we explored OXT-specific functions for these genes. By luciferase assays, we demonstrate that CREB3L1 may be a transcription factor regulating Oxt gene expression. By RNA immunoprecipitation assays and Northern blot analysis of Oxt mRNA poly(A) tails, we have found that CAPRIN2 binds to Oxt mRNA and regulates its poly(A) tail length. Moreover, in response to dehydration, Caprin2 mRNA is subjected to nuclear retention, possibly to regulate Caprin2 mRNA availability in the cytoplasm. CONCLUSION: The exploration of the spatiotemporal dynamics of Creb3l1- and Caprin2-encoded mRNAs and proteins has provided novel insights beyond the AVP-ergic system, revealing novel OXT-ergic system roles of these genes in the osmotic defence response.
Assuntos
Arginina Vasopressina , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Ocitocina , Proteínas de Ligação a RNA , Animais , Ratos , Arginina Vasopressina/genética , Arginina Vasopressina/metabolismo , Desidratação/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Ocitocina/genética , Ocitocina/metabolismo , Núcleo Hipotalâmico Paraventricular/metabolismo , RNA Mensageiro/metabolismo , Núcleo Supraóptico/metabolismo , Água/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas de Ligação a RNA/genéticaRESUMO
For decades, local bone drug delivery systems have been investigated in terms of their application in regenerative medicine. Among them, inorganic polymers based on amorphous silica have been widely explored. In this work, we combined two types of amorphous silica: bioglass and doxycycline-loaded mesoporous silica MCM-41 into the form of spherical granules (pellets) as a bifunctional bone drug delivery system. Both types of silica were obtained in a sol-gel method. The drug adsorption onto the MCM-41 was performed via adsorption from concentrated doxycycline hydrochloride solution. Pellets were obtained on a laboratory scale using the wet granulation-extrusion-spheronization method and investigated in terms of physical properties, drug release, antimicrobial activity against Staphylococcus aureus, mineralization properties in simulated body fluid, and cytotoxicity towards human osteoblasts. The obtained pellets were characterized by satisfactory mechanical properties which eliminated the risk of pellets cracking during further investigations. The biphasic drug release from pellets was observed: burst stage (44% of adsorbed drug released within the first day) followed by prolonged release with zero-order kinetics (estimated time of complete drug release was 19 days) with maintained antimicrobial activity. The progressive biomimetic apatite formation on the surface of the pellets was observed. No cytotoxic effect of pellets towards human osteoblasts was noticed.
Assuntos
Substitutos Ósseos/administração & dosagem , Substitutos Ósseos/química , Cerâmica/química , Sistemas de Liberação de Medicamentos , Dióxido de Silício/administração & dosagem , Dióxido de Silício/química , Adsorção , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Regeneração Óssea , Substitutos Ósseos/farmacocinética , Calcificação Fisiológica , Varredura Diferencial de Calorimetria , Doxiciclina/administração & dosagem , Doxiciclina/farmacocinética , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Humanos , Técnicas In Vitro , Teste de Materiais , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Medicina Regenerativa , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus/efeitos dos fármacosRESUMO
Rosa canina L. (dog rose) is a rich source of phenolic compounds that offer great hope for the prevention of chronic human diseases. Herein, wild and commercial samples of dog rose were chemically characterized with respect to their phenolic composition by liquid chromatography coupled to diode array detection and electrospray ionization tandem mass spectrometry (LC-DAD-ESI/MS). Furthermore, in vitro antioxidant properties and antibacterial activity of dog rose fruits and leaves hydromethanolic extracts and infusions were also evaluated. The results revealed that wild and commercial fruits of dog rose are similar in terms of l(+)-ascorbic acid, total phenolics (TPC), total flavonoids (TFC) and total phenolic acids (TPAC) content, individual phenolic constituents and antioxidant activity. Moreover, the fruits had lower levels of phenolic compounds and also revealed lower biological activity than the leaves. On the other hands, the highest content of TPC, TFC, TPAC, individual phenolic constituents, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity and FRAP (ferric reducing antioxidant power) were found in the leaf's infusions. They were also the only ones to show antibacterial activity. Overall, these finding confirmed usefulness of R. canina L. leaves and fruits as a rich source of bioactive phenolic compounds with potential use in food, pharmaceutical, and cosmetic industries.
Assuntos
Antioxidantes/química , Frutas/química , Fenóis/química , Extratos Vegetais/farmacologia , Folhas de Planta/química , Rosa/química , Compostos de Bifenilo/química , Cromatografia Líquida , Cosméticos , Escherichia coli/efeitos dos fármacos , Flavonoides/química , Hidroxibenzoatos/química , Testes de Sensibilidade Microbiana , Picratos/química , Pseudomonas aeruginosa/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus epidermidis/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacosRESUMO
The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.
Assuntos
Sistema Hipotálamo-Hipofisário/metabolismo , Osmorregulação/fisiologia , Neuro-Hipófise/metabolismo , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Animais , Arginina Vasopressina/sangue , Arginina Vasopressina/efeitos dos fármacos , Bumetanida/farmacologia , Desidratação/fisiopatologia , Furosemida/farmacologia , Expressão Gênica/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/citologia , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Masculino , Núcleos da Linha Média do Tálamo/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Quiasma Óptico/fisiologia , Neuro-Hipófise/citologia , Neuro-Hipófise/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Transgênicos , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Membro 1 da Família 12 de Carreador de Soluto/biossíntese , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Astragalus membranaceus Fisch. ex Bunge (syn. Astragalus mongholicus Bunge) is one of the notable medicinal and food plants. Therefore, the aim of this study was to calculate the phenolic composition and antioxidant, antimicrobial, as well as enzyme inhibitory [acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase (TYR)] activities with chemometric approaches of the hydromethanolic and water extracts of commercial A. membranaceus samples. Ten individual phenolic compounds were determined using high-performance liquid chromatography (HPLC), and only quercetin was found at a level of above 80 µg/g DW in both extracts. Moreover, the highest antioxidant activity in DPPH, FRAP, ABTS, and CUPRAC assays was found in the sample containing the roots in loose form from USA. A. membranaceus extracts displayed the inhibition zone diameters within the range from 10 to 22 mm antimicrobial activity against S. aureus, while there were no inhibition zones in any extracts in case of E. coli. The extracts of A. membranaceous showed an inhibition rate below 40% against TYR, and among tested extracts, only two samples were able to inhibit BChE with IC50 values of above 30 µg/mL. Correlation analysis showed a highly positive relationship between their phenolic composition and antioxidant activity. Concluding, the obtained results confirmed that A. membranaceus commercial samples could be an important dietary source of natural antioxidants.
RESUMO
Immunomodulatory imide drugs (IMiDs) including thalidomide, lenalidomide, and pomalidomide, can be used to induce degradation of a protein of interest that is fused to a short zinc finger (ZF) degron motif. These IMiDs, however, also induce degradation of endogenous neosubstrates, including IKZF1 and IKZF3. To improve degradation selectivity, we took a bump-and-hole approach to design and screen bumped IMiD analogs against 8380 ZF mutants. This yielded a bumped IMiD analog that induces efficient degradation of a mutant ZF degron, while not affecting other cellular proteins, including IKZF1 and IKZF3. In proof-of-concept studies, this system was applied to induce efficient degradation of TRIM28, a disease-relevant protein with no known small molecule binders. We anticipate that this system will make a valuable addition to the current arsenal of degron systems for use in target validation.
RESUMO
In the present study, the chemical composition and bioactive properties of commercially available Withania somnifera samples were evaluated. The hydromethanolic and aqueous extracts of the tested samples were analyzed in terms of phenolic compound composition, ascorbic acid content, antioxidant and antibacterial activity, and acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. Polyphenols and ascorbic acid content, as well as the antioxidant activity, were higher in the aqueous extracts than in the hydromethanolic extracts. Generally, aqueous extracts presented higher antioxidant activity than the hydromethanolic ones, especially in the case of 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay. Moreover, higher amounts of phenolic acids and flavonoids were found in the hydromethanolic extracts compared to the aqueous ones. Regarding the antibacterial properties, samples 4, 6, and 10 showed the best overall performance with growth-inhibitory activities against all the examined bacteria strains. Finally, the aqueous and hydromethanolic extracts were the most efficient extracts in terms of AChE and BChE inhibitory activities, respectively. In conclusion, our results indicate that W. somnifera possesses important bioactive properties which could be attributed to the high amounts of phenolic compounds. However, a great variability was recorded in commercially available products, suggesting significant differences in the origin of product and the processing method.
RESUMO
Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity.
RESUMO
Targeted protein degradation (TPD), induced by enforcing target proximity to an E3 ubiquitin ligase using small molecules has become an important drug discovery approach for targeting previously undruggable disease-causing proteins. However, out of over 600 E3 ligases encoded by the human genome, just over 10 E3 ligases are currently utilized for TPD. Here, using the affinity-directed protein missile (AdPROM) system, in which an anti-GFP nanobody was linked to an E3 ligase, we screened over 30 E3 ligases for their ability to degrade 4 target proteins, K-RAS, STK33, ß-catenin, and FoxP3, which were endogenously GFP-tagged. Several new E3 ligases, including CUL2 diGly receptor KLHDC2, emerged as effective degraders, suggesting that these E3 ligases can be taken forward for the development of small-molecule degraders, such as proteolysis targeting chimeras (PROTACs). As a proof of concept, we demonstrate that a KLHDC2-recruiting peptide-based PROTAC connected to chloroalkane is capable of degrading HALO-GFP protein in cells.
Assuntos
Fatores de Transcrição , beta Catenina , Humanos , beta Catenina/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteólise , Descoberta de Drogas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
BACKGROUND: Oxidative stress and dyslipidemia play a critical role in the development of cardiovascular disease (CVD). Regular intake of polyphenol-rich diets is associated with a reduced risk of CVDs. METHODS: The present study was a pilot study with 24 healthy volunteers and was designed to determine if a 12-week administration of Cistus incanus herbal tea, containing phenolic acids and flavonoids, reduces cardiovascular risk factors including oxidative stress and dyslipidemia in healthy adults. Phenolic compounds profile and antibacterial activity of Cistus incanus infusion were also measured. RESULTS: Herbal infusion led to improvement in lipid profile by increase (D4%, p = 0.033) high-density lipoprotein cholesterol concentration and decrease triglyceride (D14%, p = 0.013) concentrations. In addition, the Cistus incanus diet was associated with decreased serum concentrations of malondialdehyde (D16%, p < 0.01) and advanced oxidation protein products (D18%, p < 0.001). CONCLUSIONS: Cistus incanus administration decreases cardiovascular risk factors including oxidative stress and dyslipidemia and this action supports the idea of using Cistus incanus tea on a daily basis as an effective dietary component for prevention of atherosclerotic CVD.
Assuntos
Cistus , Chás de Ervas , Adulto , Suplementos Nutricionais , Humanos , Lipídeos , Estresse Oxidativo , Projetos Piloto , Extratos Vegetais/farmacologiaRESUMO
Targeted protein degradation is an emerging new strategy for the modulation of intracellular protein levels with applications in chemical biology and drug discovery. One approach to enable this strategy is to redirect the ubiquitin-proteasome system to mark and degrade target proteins of interest (POIs) through the use of proteolysis targeting chimeras (PROTACs). Although great progress has been made in enabling PROTACs as a platform, there are still a limited number of E3 ligases that have been employed for PROTAC design. Herein we report a novel phenotypic screening approach for the identification of E3 ligase binders. The key concept underlying this approach is the high-throughput modification of screening compounds with a chloroalkane moiety to generate HaloPROTACs in situ, which were then evaluated for their ability to degrade a GFP-HaloTag fusion protein in a cellular context. As proof of concept, we demonstrated that we could generate and detect functional HaloPROTACs in situ, using a validated Von Hippel-Lindau (VHL) binder that successfully degraded the GFP-HaloTag fusion protein in living cells. We then used this method to prepare and screen a library of approximately 2000 prospective E3 ligase-recruiting molecules.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteólise/efeitos dos fármacos , Humanos , Ligação Proteica , Bibliotecas de Moléculas Pequenas , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
For decades, bone drug delivery systems dedicated for osteomyelitis treatment have been investigated as bifunctional materials that exhibit prolonged drug release and mineralization potential. Herein, composite-type pellets based on cefazolin-loaded amino-modified mesoporous silica SBA-15 and microwave-assisted hydroxyapatite were investigated as potential bone drug delivery system in vitro. Pellets were obtained by granulation, extrusion and spheronization methods in laboratory scale and studied in terms of physical properties, drug release, mineralization potential, antimicrobial activity and cytotoxicity towards human osteoblasts. The obtained pellets were characterized for hardness and friability which indicated the pellets durability during further investigations. Prolonged (5-day) release of cefazolin from pellets was observed. The pellets exhibited mineralization potential in simulated body fluid, i.e., a continuous layer of bone-like apatite was formed on the surface of pellets after 28 days of incubation. An antimicrobial assay of pellets revealed an antibacterial effect against Staphylococcus aureus strain during 6 days. No cytotoxic effects of pellets towards human osteoblasts were observed. The obtained results proved that proposed pellets appear to have potential applications as bone drug delivery systems.
Assuntos
Antibacterianos/química , Cefazolina/química , Portadores de Fármacos , Dióxido de Silício/química , Antibacterianos/administração & dosagem , Antibacterianos/toxicidade , Cefazolina/administração & dosagem , Cefazolina/toxicidade , Linhagem Celular , Preparações de Ação Retardada , Composição de Medicamentos , Implantes de Medicamento , Liberação Controlada de Fármacos , Durapatita/química , Dureza , Humanos , Cinética , Osteoblastos/efeitos dos fármacos , Osteomielite/tratamento farmacológico , Osteomielite/microbiologia , Porosidade , Dióxido de Silício/toxicidade , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Propriedades de SuperfícieRESUMO
K-RAS is known as the most frequently mutated oncogene. However, the development of conventional K-RAS inhibitors has been extremely challenging, with a mutation-specific inhibitor reaching clinical trials only recently. Targeted proteolysis has emerged as a new modality in drug discovery to tackle undruggable targets. Our laboratory has developed a system for targeted proteolysis using peptidic high-affinity binders, called "AdPROM." Here, we used CRISPR/Cas9 technology to knock in a GFP tag on the native K-RAS gene in A549 adenocarcinoma (A549GFPKRAS) cells and constructed AdPROMs containing high-affinity GFP or H/K-RAS binders. Expression of GFP-targeting AdPROM in A549GFPKRAS led to robust proteasomal degradation of endogenous GFP-K-RAS, while expression of anti-HRAS-targeting AdPROM in different cell lines resulted in the degradation of both GFP-tagged and untagged K-RAS, and untagged H-RAS. Our findings imply that endogenous RAS proteins can be targeted for proteolysis, supporting the idea of an alternative therapeutic approach to these undruggable targets.
Assuntos
Proteólise , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células A549 , Marcadores de Afinidade , Sistemas CRISPR-Cas/genética , Linhagem Celular , Proliferação de Células , Técnicas de Introdução de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia de Fluorescência , Peptídeos/química , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The Bcl-2 family of proteins, such as Bcl-xL and Bcl-2, play key roles in cancer cell survival. Structural studies of Bcl-xL formed the foundation for the development of the first Bcl-2 family inhibitors and FDA approved drugs. Recently, Proteolysis Targeting Chimeras (PROTACs) that degrade Bcl-xL have been proposed as a therapeutic modality with the potential to enhance potency and reduce toxicity versus antagonists. However, no ternary complex structures of Bcl-xL with a PROTAC and an E3 ligase have been successfully determined to guide this approach. Herein, we report the design, characterization, and X-ray structure of a VHL E3 ligase-recruiting Bcl-xL PROTAC degrader. The 1.9 Å heterotetrameric structure, composed of (ElonginB:ElonginC:VHL):PROTAC:Bcl-xL, reveals an extensive network of neo-interactions, between the E3 ligase and the target protein, and between noncognate parts of the PROTAC and partner proteins. This work illustrates the challenges associated with the rational design of bifunctional molecules where interactions involve composite interfaces.
Assuntos
Benzotiazóis/metabolismo , Isoquinolinas/metabolismo , Oligopeptídeos/metabolismo , Proteólise/efeitos dos fármacos , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Proteína bcl-X/antagonistas & inibidores , Benzotiazóis/química , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Humanos , Isoquinolinas/química , Isoquinolinas/farmacologia , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Ligação Proteica , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
Ammonia-induced swelling of astrocytes is a primary cause of brain edema associated with acute hepatic encephalopathy. Previous studies have shown that ammonia transiently increases cGMP in brain in vivo and in cultured astrocytes in vitro. We hypothesized that protein kinase G (PKG), an enzyme activated by cGMP and implicated in regulation of cell shape, size, and/or volume in peripheral and CNS cells, may play a role in the ammonia-induced astrocytic volume increase. Treatment of cultured rat cortical astrocytes with 1 or 5 mM NH4Cl (ammonia) for 24 h increased their cell volume by 50% and 80% above control, respectively, as measured by confocal imaging followed by 3D computational analysis. A cGMP analog, 8-(4-chlorophenylthio)-cGMP, increased the cell volume in control cells and potentiated the increase in 1 mM ammonia-treated cells. A soluble guanylate cyclase inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) abrogated, and a PKG inhibitor [8-(4-chlorophenylthio)-cGMP-thioate, Rp-isomer] dose-dependently reduced the cell volume-increasing effect of 5 mM ammonia. The results suggest that (i) PKG may play a permissive role in ammonia-induced astrocytic swelling and (ii) elevation of brain cGMP associated with acute exposure to ammonia in vivo may aggravate the ensuing brain edema.
Assuntos
Amônia/toxicidade , Astrócitos/efeitos dos fármacos , Astrócitos/enzimologia , Proteínas Quinases Dependentes de GMP Cíclico/fisiologia , Animais , Astrócitos/patologia , Edema Encefálico/patologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Tamanho Celular , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes , Guanilato Ciclase/antagonistas & inibidores , Microscopia Confocal , Nimodipina/farmacologia , Ratos , Ratos Wistar , Tionucleotídeos/farmacologiaRESUMO
Bone tissue inflammation, osteomyelitis, is commonly caused by bacterial invasion and requires prolonged antibiotic therapy for weeks or months. Thus, the aim of this study was to develop novel silica-polymer local bone antibiotic delivery systems characterized by a sustained release of ciprofloxacin (CIP) which remain active against Staphylococcus aureus for a few weeks, and do not have a toxic effect towards human osteoblasts. Four formulations composed of ethylcellulose (EC), polydimethylsiloxane (PDMS), freeze-dried CIP, and CIP-adsorbed mesoporous silica materials (MCM-41-CIP) were prepared via solvent-evaporation blending method. All obtained composites were characterized in terms of molecular structure, morphological, and structural properties by using Fourier Transform Infrared Spectroscopy (FTIR), scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy (SEM/EDX), and X-ray diffraction (XRD), thermal stability by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC), and in vitro antibiotic release. The antibacterial activity against Staphylococcus aureus (ATCC 6538) as well as the in vitro cytocompatibility to human osteoblasts of obtained composites were also examined. Physicochemical results confirmed the presence of particular components (FTIR), formation of continuous polymer phase onto the surface of freeze-dried CIP or MCM-41-CIP (SEM/EDX), and semi-crystalline (composites containing freeze-dried CIP) or amorphous (composites containing MCM-41-CIP) structure (XRD). TGA and DSC analysis indicated the high thermal stability of CIP adsorbed onto the MCM-41, and higher after MCM-41-CIP coating with polymer blend. The release study revealed the significant reduction in initial burst of CIP for the composites which contained MCM-41-CIP instead of freeze-dried CIP. These composites were also characterized by the 30-day activity against S. aureus and the highest cytocompatibility to human osteoblasts in vitro.
RESUMO
A series of novel fluoroquinolone-Safirinium dye hybrids was synthesized by means of tandem Mannich-electrophilic amination reactions from profluorophoric isoxazolones and antibiotics bearing a secondary amino group at position 7 of the quinoline ring. The obtained fluorescent spiro fused conjugates incorporating quaternary nitrogen atoms were characterized by 1H NMR, IR, MS, and elemental analysis. All the synthetic analogues (3a-h and 4a-h) were evaluated for their in vitro antimicrobial, bactericidal, and antibiofilm activities against a panel of Gram positive and Gram-negative pathogenic bacteria. The most active Safirinium Q derivatives of lomefloxacin (4d) and ciprofloxacin (4e) exhibited molar-based antibacterial activities comparable to the unmodified drugs and displayed considerable inhibitory potencies in E. coli DNA gyrase supercoiling assays with IC50 values in the low micromolar range. Zwiterionic hybrids were noticeably less lipophilic than the parent quinolones in micellar electrokinetic chromatography (MECK) experiments. The tests performed in the presence of phenylalanine-arginine ß-naphthylamide (PAßN) or carbonyl cyanide m-chlorophenylhydrazone (CCCP) revealed that the conjugates are to some extent subject to bacterial efflux and cellular accumulation, respectively. Moreover, the hybrids did not exhibit notable cytotoxicity towards the HEK 293 control cell line and demonstrated low propensity for resistance development, as exemplified for compounds 3g and 4b. Finally, molecular docking experiments revealed that the synthesized compounds were able to bind in the fluoroquinolone-binding mode at S. aureus DNA gyrase and S. pneumoniae topoisomerase IV active sites.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Compostos de Amônio Quaternário/farmacologia , Quinolonas/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerase IV/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Células HEK293 , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Compostos de Amônio Quaternário/síntese química , Compostos de Amônio Quaternário/química , Quinolonas/química , Relação Estrutura-AtividadeRESUMO
Recently we reported a decrease of C-type natriuretic peptide (CNP)-dependent, natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP (cGMP) synthesis in a non-neuronal compartment of cerebral cortical slices of hyperammonemic rats [Zielinska, M., Fresko, I., Konopacka, A., Felipo, V., Albrecht, J., 2007. Hyperammonemia inhibits the natriuretic peptide receptor 2 (NPR2)-mediated cyclic GMP synthesis in the astrocytic compartment of rat cerebral cortex slices. Neurotoxicology 28, 1260-1263]. Here we accounted for the possible involvement of cerebral capillary endothelial cells in this response by measuring the effect of ammonia on the CNP-mediated cGMP formation and intracellular calcium ([Ca2+]i) accumulation in a rat cerebral endothelial cell line (RBE-4). We first established that stimulation of cGMP synthesis in RBE-4 cells was coupled to protein kinase G (PKG)-mediated Ca2+ influx from the medium which was inhibited by an L-type channel blocker nimodipine. Ammonia treatment (1h, 5mM NH4Cl) evoked a substantial decrease of CNP-stimulated cGMP synthesis which was related to a decreased binding of CNP to NPR2 receptors, and depressed the CNP-dependent [Ca2+]i accumulation in these cells. Ammonia also abolished the CNP-dependent Ca2+ accumulation in the absence of Na+. In cells incubated with ammonia in the absence of Ca2+ a slight CNP-dependent increase of [Ca2+]i was observed, most likely representing Ca2+ release from intracellular stores. Depression of CNP-dependent cGMP-mediated [Ca2+]i accumulation may contribute to cerebral vascular endothelial dysfunction associated with hyperammonemia or hepatic encephalopathy.
Assuntos
Amônia/metabolismo , Cálcio/metabolismo , Artérias Cerebrais/metabolismo , GMP Cíclico/biossíntese , Células Endoteliais/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Amônia/toxicidade , Compostos de Anilina , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Artérias Cerebrais/fisiopatologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Proteínas Quinases Dependentes de GMP Cíclico/efeitos dos fármacos , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Células Endoteliais/efeitos dos fármacos , Encefalopatia Hepática/metabolismo , Encefalopatia Hepática/fisiopatologia , Hiperamonemia/metabolismo , Hiperamonemia/fisiopatologia , Peptídeo Natriurético Tipo C/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Receptores do Fator Natriurético Atrial/efeitos dos fármacos , Receptores do Fator Natriurético Atrial/metabolismo , XantenosRESUMO
Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.
Assuntos
Calmodulina/química , Indóis/química , Oximas/química , Pirazóis/química , Pirimidinas/química , Riluzol/química , Canais de Potássio Ativados por Cálcio de Condutância Baixa/química , Regulação Alostérica , Motivos de Aminoácidos , Anticonvulsivantes/química , Anticonvulsivantes/metabolismo , Sítios de Ligação , Calmodulina/genética , Calmodulina/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Indóis/metabolismo , Modelos Moleculares , Oximas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Domínios e Motivos de Interação entre Proteínas , Pirazóis/metabolismo , Pirimidinas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Riluzol/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismoRESUMO
The decrease of cyclic GMP (cGMP) level in the brain, contributing to cognitive and memory deficit in hyperammonemia (HA), has been attributed to the interference of ammonia with the NMDA/nitric oxide/soluble guanylate cyclase (GC)/cGMP pathway in neurons. The present study tested the hypotheses that (a) HA also affects cGMP synthesis elicited by stimulation of the natriuretic peptide receptor 2 (NPR-2) with its natural ligand, C-type natriuretic peptide (CNP) and (b) the latter effect may involve astrocytes, the ammonia-sensitive cells. In the cerebral cortical slices of control rats, CNP stimulated cGMP synthesis in a degree comparable to the NO donor, S-nitroso-N-acetylpenicillamine (SNAP) used at an optimal concentration. Fluoroacetate (FA), a metabolic inhibitor specifically affecting astrocytic mitochondria, inhibited the CNP-dependent cGMP synthesis by about 50%. Ammonium acetate-induced HA decreased by 68% the CNP-dependent cGMP generation in slices incubated in the absence of FA. In slices incubated in the presence of FA, cGMP synthesis in slices derived from HA rats did not differ from that in control slices. The results indicate that HA inhibits CNP-dependent cGMP synthesis in the FA-vulnerable, astrocytic compartment, but not in the FA-resistant compartment(s) of the brain. HA did not affect the expression of NPR-2 mRNA in the cerebral cortex tissue as tested using real-time PCR, indicating that the effect of ammonia involves as yet unidentified events occurring posttranscriptionally. Deregulation of NPR-2 function in astrocytes by ammonia may contribute to neurophysiological symptoms of HA.