RESUMO
Cognitive impairment represents one of the core features of schizophrenia. Prolyl Oligopeptidase (POP) inhibition is an emerging strategy for compensating cognitive deficits in hypoglutamatergic states such as schizophrenia, although little is known about how POP inhibitors exert their pharmacological activity. The mitochondrial and nuclear protein Prohibitin 2 (PHB2) could be dysregulated in schizophrenia. However, altered PHB2 levels in schizophrenia linked to N-methyl-D-aspartate receptor (NMDAR) activity and cognitive deficits are still unknown. To shed light on this, we measured the PHB2 levels by immunoblot in a postmortem dorsolateral prefrontal cortex (DLPFC) of schizophrenia subjects, in the frontal pole of mice treated with the NMDAR antagonists phencyclidine and dizocilpine, and in rat cortical astrocytes and neurons treated with dizocilpine. Mice and cells were treated in combination with the POP inhibitor IPR19. The PHB2 levels were also analyzed by immunocytochemistry in rat neurons. The PHB2 levels increased in DLPFC in cases of chronic schizophrenia and were associated with cognitive impairments. NMDAR antagonists increased PHB2 levels in the frontal pole of mice and in rat astrocytes and neurons. High levels of PHB2 were found in the nucleus and cytoplasm of neurons upon NMDAR inhibition. IPR19 restored PHB2 levels in the acute NMDAR inhibition. These results show that IPR19 restores the upregulation of PHB2 in an acute NMDAR hypoactivity stage suggesting that the modulation of PHB2 could compensate NMDAR-dependent cognitive impairments in schizophrenia.
Assuntos
Disfunção Cognitiva , Transtornos Psicóticos , Esquizofrenia , Animais , Ratos , Cognição , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/etiologia , Maleato de Dizocilpina/farmacologia , Proibitinas , Prolil Oligopeptidases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/tratamento farmacológico , Esquizofrenia/metabolismoRESUMO
Matrix metalloproteinases (MMPs), a family of zinc-containing endopeptidases involved in the degradation of the extracellular matrix, make a major contribution to the progression of a vast number of diseases, such cancer or epilepsy. Although several MMP inhibitors (MMPi) have been developed to date for the treatment of cancer, they have all failed in clinical trials due to lack of efficacy and, most importantly, the presence of severe side effects. The latter can be explained by their lack of selectivity of these inhibitors. In this regard, MMPs' family members have a high structural homology, which challenge the development of selective inhibitors for a specific MMP. Here, we have used in silico calculations and in vitro data to design MMPi that selectively target gelatinases (MMP-2 and MMP-9) and have the capacity to cross the blood-brain barrier. Following this approach, we obtained compound 40 that shows high proteolytic stability and low cytotoxicity. This compound may be of particular interest for the treatment of central nervous diseases such epilepsy or Alzheimer's disease, where gelatinase activity is increased. Our data show the specificity of compound 40 for recombinant MMP-9 and MMP-2 and endogenous MMP-9 from rat hippocampal cell cultures, and reveals its permeability across the blood-brain barrier in vivo.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Desenho de Fármacos , Gelatinases/antagonistas & inibidores , Ácidos Hidroxâmicos/farmacologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Gelatinases/metabolismo , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Estrutura Molecular , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
The blood-brain barrier (BBB) is a formidable physical and enzymatic barrier that tightly controls the passage of molecules from the blood to the brain. In fact, less than 2 % of all potential neurotherapeutics are able to cross it. Here, by applying the retro-enantio approach to a peptide that targets the transferrin receptor, a full protease-resistant peptide with the capacity to act as a BBB shuttle was obtained and thus enabled the transport of a variety of cargos into the central nervous system.
Assuntos
Barreira Hematoencefálica/metabolismo , Peptídeos/síntese química , Peptídeos/farmacocinética , Animais , Transporte Biológico , Bovinos , Fármacos do Sistema Nervoso Central/farmacocinética , Técnicas de Cocultura , Células Endoteliais/metabolismo , Camundongos , Peptídeo Hidrolases/química , Permeabilidade , Ratos , Receptores da Transferrina/efeitos dos fármacos , EstereoisomerismoRESUMO
Less than 2% of all potential neurotherapeutics cross the blood-brain barrier (BBB). Here, we sought to build a construct with the capacity to cross this barrier, to behave as a chemical delivery system, and, once inside the central nervous system, to be transformed and then act as an enzyme inhibitor. With all this in mind, here, we describe the entire process to obtain such a compound, from the initial candidate selection to preparation of the compound library and posterior evaluation and final selection of the most promising candidates in terms of selectivity, serum stability, and BBB-transport.
Assuntos
Barreira Hematoencefálica , Transporte Biológico , HumanosRESUMO
Oncogenic RAS proteins are important for driving tumour formation, and for maintenance of the transformed phenotype, and thus their relevance as a cancer therapeutic target is undeniable. We focused here on obtaining peptidomimetics, which have good pharmacological properties, to block Ras-effector interaction. Computational analysis was used to identify hot spots of RAS relevant for these interactions and to screen a library of peptidomimetics. Nine compounds were synthesized and assayed for their activity as RAS inhibitors in cultured cells. Most of them induced a reduction in ERK and AKT activation by EGF, a marker of RAS activity. The most potent inhibitor disrupted Raf and PI3K interaction with oncogenic KRAS, corroborating its mechanism of action as an inhibitor of protein-protein interactions, and thus validating our computational methodology. Most interestingly, improvement of one of the compounds allowed us to obtain a peptidomimetic that decreased the survival of pancreatic cancer cell lines harbouring oncogenic KRAS.
Assuntos
Neoplasias Pancreáticas , Peptidomiméticos , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Humanos , Neoplasias Pancreáticas/metabolismo , Peptidomiméticos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Transdução de Sinais/genéticaRESUMO
Matrix metalloproteinases (MMPs) are endopeptidases responsible for the cleavage of intra- and extracellular proteins. Several brain MMPs have been implicated in neurological disorders including epilepsy. We recently showed that the novel gelatinase inhibitor ACT-03 has disease-modifying effects in models of epilepsy. Here, we studied its effects on neuroinflammation and blood-brain barrier (BBB) integrity. Using the rapid kindling rat model of epilepsy, we examined whether ACT-03 affected astro- and microgliosis in the brain using immunohistochemistry. Cellular and molecular alterations were further studied in vitro using human fetal astrocyte and brain endothelial cell (hCMEC/D3) cultures, with a focus on neuroinflammatory markers as well as on barrier permeability using an endothelial and astrocyte co-culture model. We observed less astro- and microgliosis in the brains of kindled animals treated with ACT-03 compared to control vehicle-treated animals. In vitro, ACT-03 treatment attenuated stimulation-induced mRNA expression of several pro-inflammatory factors in human fetal astrocytes and brain endothelial cells, as well as a loss of barrier integrity in endothelial and astrocyte co-cultures. Since ACT-03 has disease-modifying effects in epilepsy models, possibly via limiting gliosis, inflammation, and barrier integrity loss, it is of interest to further evaluate its effects in a clinical trial.
RESUMO
G protein-coupled receptors transduce signals through heterotrimeric G protein Gα and Gßγ subunits, both of which interact with downstream effectors to regulate cell function. Gßγ signaling has been implicated in the pathophysiology of several diseases, suggesting that Gßγ could be an important pharmaceutical target. Previously, we used a combination of virtual and manual screening to find small molecules that bind to a protein-protein interaction "hot spot" on Gßγ and block regulation of physiological effectors. One of the most potent and effective compounds from this screen was selenocystamine. In this study, we investigated the mechanism of action of selenocystamine and found that selenocysteamine forms a covalent complex with Gßγ by a reversible redox mechanism. Mass spectrometry and site-directed mutagenesis suggest that selenocysteamine preferentially modifies GßCys204, but also a second undefined site. The high potency of selenocystamine in Gßγ inhibition seems to arise from both high reactivity of the diselenide group and binding to a specific site on Gß. Using structural information about the "hot spot," we developed a strategy to selectively target redox reversible compounds to a specific site on Gßγ using peptide carriers such as SIGCAFKILGY(-cysteamine) [SIGC(-cysteamine)]. Mass spectrometry and site-directed mutagenesis indicate that SIGC(-cysteamine) specifically and efficiently leads to cysteamine (half-cystamine) modification of a single site on Gß, likely GßCys204, and inhibits Gßγ more than a hundred times more potently than cystamine. These data support the concept that covalent modifiers can be specifically targeted to the Gßγ "hot spot" through rational incorporation into molecules that noncovalently bind to Gßγ.
Assuntos
Cistamina/análogos & derivados , Desenho de Fármacos , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Compostos Organosselênicos/química , Sequência de Aminoácidos , Cistamina/química , Cistamina/farmacologia , Quinase 2 de Receptor Acoplado a Proteína G/antagonistas & inibidores , Quinase 2 de Receptor Acoplado a Proteína G/química , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/antagonistas & inibidores , Subunidades gama da Proteína de Ligação ao GTP/antagonistas & inibidores , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Compostos Organosselênicos/farmacologia , Oxirredução/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologiaRESUMO
Matrix metalloproteinases (MMPs) are synthesized by neurons and glia and released into the extracellular space, where they act as modulators of neuroplasticity and neuroinflammatory agents. Development of epilepsy (epileptogenesis) is associated with increased expression of MMPs, and therefore, they may represent potential therapeutic drug targets. Using quantitative PCR (qPCR) and immunohistochemistry, we studied the expression of MMPs and their endogenous inhibitors tissue inhibitors of metalloproteinases (TIMPs) in patients with status epilepticus (SE) or temporal lobe epilepsy (TLE) and in a rat TLE model. Furthermore, we tested the MMP2/9 inhibitor IPR-179 in the rapid-kindling rat model and in the intrahippocampal kainic acid mouse model. In both human and experimental epilepsy, MMP and TIMP expression were persistently dysregulated in the hippocampus compared with in controls. IPR-179 treatment reduced seizure severity in the rapid-kindling model and reduced the number of spontaneous seizures in the kainic acid model (during and up to 7 weeks after delivery) without side effects while improving cognitive behavior. Moreover, our data suggest that IPR-179 prevented an MMP2/9-dependent switch-off normally restraining network excitability during the activity period. Since increased MMP expression is a prominent hallmark of the human epileptogenic brain and the MMP inhibitor IPR-179 exhibits antiseizure and antiepileptogenic effects in rodent epilepsy models and attenuates seizure-induced cognitive decline, it deserves further investigation in clinical trials.
Assuntos
Encéfalo/enzimologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Inibidores de Metaloproteinases de Matriz/farmacologia , Estado Epiléptico/tratamento farmacológico , Animais , Encéfalo/patologia , Epilepsia do Lobo Temporal/enzimologia , Epilepsia do Lobo Temporal/patologia , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Ratos , Ratos Sprague-Dawley , Estado Epiléptico/enzimologia , Estado Epiléptico/patologiaRESUMO
In recent decades, peptide blood-brain barrier shuttles have emerged as a promising solution for brain drugs that are not able to enter this organ. The research and development of these compounds involve the use of in vitro cell-based models of the BBB. Nevertheless, peptide transport quantification implies the use of large amounts of peptide (upper micromolar range for RP-HPLC-PDA) or of derivatives (e.g. fluorophore or quantum-dot attachment, radiolabeling) in the donor compartment in order to enhance the detection of these molecules in the acceptor well, although their structure is highly modified. Therefore, these methodologies either hamper the use of low peptide concentrations, thus hindering mechanistic studies, or do not allow the use of the unmodified peptide. Here we successfully applied a MALDI-TOF MS methodology for transport quantification in an in vitro BBB cell-based model. A light version of the acetylated peptide was evaluated, and the transport was subsequently quantified using a heavy internal standard (isotopically acetylated). We propose that this MALDI-TOF MS approach could also be applied to study the transport across other biological barriers using the appropriate in vitro transport models (e.g. Caco-2, PAMPA).
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Peptídeos/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Bovinos , Linhagem Celular , Humanos , Peptídeos/química , Transporte Proteico/efeitos dos fármacos , Pontos Quânticos/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima.
Assuntos
Lipoproteínas LDL/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Peptídeos/metabolismo , Proteínas de Artrópodes/sangue , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Oligopeptídeos/sangue , Fosfolipases A2/metabolismo , Fosfolipídeos/química , Ligação Proteica , Esfingomielina Fosfodiesterase/química , Eletricidade EstáticaRESUMO
Prolyl oligopeptidase is a cytosolic serine peptidase that hydrolyzes proline-containing peptides at the carboxy terminus of proline residues. It has been associated with schizophrenia, bipolar affective disorder, and related neuropsychiatric disorders and therefore may have important clinical implications. In a previous work, we used (19)F NMR to search for new prolyl oligopeptidase inhibitors from a library of traditional Chinese medicine plant extracts, and identified several extracts as powerful inhibitors of this peptidase. Here, the flavonoid baicalin was isolated as the active component of an extract of Scutellaria baicalensis roots having prolyl oligopeptidase inhibitory activity. Baicalin inhibited prolyl oligopeptidase in a dose-dependent manner. Inhibition experiments using baicalin analogs showed that the sugar moiety was not necessary for activity. The IC(50)s of baicalin and its aglycone derivative baicalein were rather similar, showing that the sugar moiety was not involved in the interaction of baicalin with POP. These results were confirmed by saturation transfer difference NMR experiments. To further understand the absorption and transport mechanisms of baicalin and baicalein, we evaluated their transport in vitro through the gastrointestinal tract and the blood-brain barrier using a Parallel Artificial Membrane Permeability Assay. The molecule which potentially crosses both barriers was identified as baicalein, the aglycone moiety of baicalin. Our results show that baicalin is a new prodrug able to inhibit prolyl oligopeptidase. As baicalin is a natural compound with a long history of safe administration to humans, it is a highly attractive base from which to develop new treatments for schizophrenia, bipolar affective disorder, and related neuropsychiatric diseases.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flavonoides/química , Flavonoides/farmacologia , Serina Endopeptidases/metabolismo , Relação Dose-Resposta a Droga , Flavanonas/química , Flavanonas/farmacologia , Prolil Oligopeptidases , Relação Estrutura-AtividadeRESUMO
Prolyl oligopeptidase (POP), a serine protease highly expressed in the brain, has recently emerged as an enticing therapeutic target for the treatment of cognitive and neurodegenerative disorders. However, most reported inhibitors suffer from short duration of action, poor protease selectivity, and low blood-brain barrier (BBB) permeability, which altogether limit their potential as drugs. Here, we describe the structure-based design of the first irreversible, selective, and brain-permeable POP inhibitors. At low-nanomolar concentrations, these covalent peptidomimetics produce a fast, specific, and sustained inactivation of POP, both in vitro and in human cells. More importantly, they are >1,000-fold selective against two family-related proteases (DPPIV and FAP) and display high BBB permeability, as shown in both lipid membranes and MDCK cells.
Assuntos
Fluoretos/química , Fluoretos/farmacologia , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Animais , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Cães , Descoberta de Drogas , Fluoretos/farmacocinética , Humanos , Células Madin Darby de Rim Canino , Modelos Moleculares , Peptidomiméticos/farmacocinética , Permeabilidade , Prolil Oligopeptidases , Inibidores de Serina Proteinase/farmacocinéticaRESUMO
Low effectiveness and resistance to treatments are commonplace in disorders of the central nervous system (CNS). These issues concern mainly the blood-brain barrier (BBB), which preserves homeostasis in the brain and protects this organ from toxic molecules and biohazards by regulating transport through it. BBB shuttles-short peptides able to cross the BBB-are being developed to help therapeutics to cross this barrier. BBB shuttles can be discovered by massive exploration of chemical diversity (e.g. computational means, phage display) or rational design (e.g. derivatives from a known peptide/protein able to cross). Here we present the selection of a peptide shuttle (HAI) from several candidates and the subsequent in-depth in vitro and in vivo study of this molecule. In order to explore the chemical diversity of HAI and enhance its biostability, and thereby its bioactivity, we explored two new protease-resistant versions of HAI (i.e. the retro-D-version, and a version that was N-methylated at the most sensitive sites to enzymatic cleavage). Our results show that, while both versions of HAI are resistant to proteases, the retro-D-approach preserved better transport properties.
Assuntos
Barreira Hematoencefálica/metabolismo , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/farmacocinética , Receptores da Transferrina/análise , Animais , Peptídeos Penetradores de Células/química , Sistemas de Liberação de Medicamentos , Desenho de Fármacos , Estabilidade de Medicamentos , Humanos , Peptídeo Hidrolases/metabolismo , Permeabilidade , RatosRESUMO
Cognitive deficits are considered a key feature of schizophrenia, and they usually precede the onset of the illness and continue after psychotic symptoms appear. Current antipsychotic drugs have little or no effect on the cognitive deficits of this disorder. Prolyl oligopeptidase (POP) is an 81-kDa monomeric serine protease that is expressed in brain and other tissues. POP inhibitors have shown neuroprotective, anti-amnesic and cognition-enhancing properties. Here we studied the potential of IPR19, a new POP inhibitor, for the treatment of the cognitive symptoms related to schizophrenia. The efficacy of the inhibitor was evaluated in mouse models based on subchronic phencyclidine and acute dizocilpine administration, and in adult offspring from mothers with immune reaction induced by polyinosinic:polycytidylic acid administration during pregnancy. Acute IPR19 administration (5mg/kg, i.p.) reversed the cognitive performance deficits of the three mouse models in the novel object recognition test, T-maze, and eight-arm radial maze. The compound also ameliorates deficits of the prepulse inhibition response. The in vitro inhibitory efficacy and selectivity, brain penetration and exposure time after injection of IPR19 were also addressed. Our results indicate that the inhibition of POP using IPR19 may offer a promising strategy to develop drugs to ameliorate the cognitive deficits of schizophrenia.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Prolina/análogos & derivados , Psicotrópicos/farmacologia , Esquizofrenia/tratamento farmacológico , Psicologia do Esquizofrênico , Animais , Linhagem Celular Tumoral , Cognição/efeitos dos fármacos , Cognição/fisiologia , Transtornos Cognitivos/enzimologia , Transtornos Cognitivos/etiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Atividade Motora/fisiologia , Poli I-C , Inibição Pré-Pulso/efeitos dos fármacos , Inibição Pré-Pulso/fisiologia , Prolina/química , Prolina/farmacocinética , Prolina/farmacologia , Prolina/toxicidade , Prolil Oligopeptidases , Psicotrópicos/química , Psicotrópicos/farmacocinética , Psicotrópicos/toxicidade , Reconhecimento Psicológico/efeitos dos fármacos , Reconhecimento Psicológico/fisiologia , Esquizofrenia/complicações , Esquizofrenia/enzimologia , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacocinética , Inibidores de Serina Proteinase/farmacologia , Inibidores de Serina Proteinase/toxicidadeRESUMO
Reduced glutamatergic activity and energy metabolism in the dorsolateral prefrontal cortex (DLPFC) have been described in schizophrenia. Glycogenolysis in astrocytes is responsible for providing neurons with lactate as a transient energy supply helping to couple glutamatergic neurotransmission and glucose utilization in the brain. This mechanism could be disrupted in schizophrenia. The aim of this study was to explore whether the protein levels of the astrocyte isoform of glycogen phosphorylase (PYGM), key enzyme of glycogenolysis, and the isoform A of Ras-related C3 botulinum toxin substrate 1 (RAC1), a kinase that regulates PYGM activity, are altered in the postmortem DLPFC of chronic schizophrenia patients (n=23) and matched controls (n=23). We also aimed to test NMDAR blockade effect on these proteins in the mouse cortex and cortical astrocytes and antipsychotic treatments in rats. Here we report a reduction in PYGM and RAC1 protein levels in the DLPFC in schizophrenia. We found that treatment with the NMDAR antagonist dizocilpine in mice as a model of psychosis increased PYGM and reduced RAC1 protein levels. The same result was observed in rat cortical astroglial-enriched cultures. 21-day haloperidol treatment increased PYGM levels in rats. These results show that PYGM and RAC1 are altered in the DLPFC in chronic schizophrenia and are controlled by NMDA signalling in the rodent cortex and cortical astrocytes suggesting an altered NMDA-dependent glycogenolysis in astrocytes in schizophrenia. Together, this study provides evidence of a NMDA-dependent transient local energy deficit in neuron-glia crosstalk in schizophrenia, contributing to energy deficits of the disorder.
Assuntos
Astrócitos/enzimologia , Glicogênio Fosforilase/metabolismo , Córtex Pré-Frontal/enzimologia , Esquizofrenia/enzimologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Idoso , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Astrócitos/efeitos dos fármacos , Células Cultivadas , Doença Crônica , Estudos de Coortes , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Haloperidol/farmacologia , Humanos , Isoenzimas , Masculino , Camundongos Endogâmicos C57BL , Córtex Pré-Frontal/efeitos dos fármacos , Distribuição Aleatória , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Esquizofrenia/tratamento farmacológicoRESUMO
Altered levels of transcription factor specificity protein 4 (SP4) and 1 (SP1) in the cerebellum, prefrontal cortex and/or lymphocytes have been reported in severe psychiatric disorders, including early psychosis, bipolar disorder, and chronic schizophrenia subjects who have undergone long-term antipsychotic treatments. SP4 transgenic mice show altered hippocampal-dependent psychotic-like behaviours and altered development of hippocampal dentate gyrus. Moreover, NMDAR activity regulates SP4 function. The aim of this study was to investigate SP4 and SP1 expression levels in the hippocampus in schizophrenia, and the possible effect of antipsychotics and NMDAR blockade on SP protein levels in rodent hippocampus. We analysed SP4 and SP1 expression levels in the postmortem hippocampus of chronic schizophrenia (n = 14) and control (n = 11) subjects by immunoblot and quantitative RT-PCR. We tested the effect of NMDAR blockade on SP factors in the hippocampus of mouse treated with an acute dose of MK801. We also investigated the effect of subacute treatments with haloperidol and clozapine on SP protein levels in the rat hippocampus. We report that SP4 protein and both SP4 and SP1 mRNA expression levels are significantly increased in the hippocampus in chronic schizophrenia. Likewise, acute treatment with MK801 increased both SP4 and SP1 protein levels in mouse hippocampus. In contrast, subacute treatment with haloperidol and clozapine did not significantly alter SP protein levels in rat hippocampus. These results suggest that SP4 and SP1 upregulation may be part of the mechanisms deregulated downstream of glutamate signalling pathways in schizophrenia and might be contributing to the hippocampal-dependent cognitive deficits of the disorder.
Assuntos
Regulação da Expressão Gênica/fisiologia , Hipocampo/metabolismo , Esquizofrenia/patologia , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp4/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Autopsia , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Ratos , Análise de Regressão , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp4/genética , Estatísticas não ParamétricasRESUMO
Schizophrenia is a serious life-long disease that affects a significant part of the adult population. Although there is considerably effective medication for the positive symptoms of the disease, none are available for the associated cognitive deficits. These deficits are a core feature of schizophrenia, and they severely impair the functionality and social integration of patients. POP is a promising target for the treatment of the cognitive deficits of schizophrenia. Inhibitors of this peptidase show cognition-enhancing properties, act through a complex mechanism and have suitable pharmacological properties. Nevertheless, several studies must be carried out in order to improve the design and clinical evaluation of these substances. Permeability to the brain, appropriate animal models and suitable indications are the main issues that must be addressed. However, current information supports the potential of POP as an interesting drug target for the treatment of the cognitive deficits related to schizophrenia.
Assuntos
Esquizofrenia/tratamento farmacológico , Esquizofrenia/enzimologia , Serina Proteases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/uso terapêutico , Animais , Descoberta de Drogas/métodos , Humanos , Terapia de Alvo Molecular/métodos , Peptídeos/química , Peptídeos/farmacologia , Peptídeos/uso terapêutico , Peptidomiméticos/química , Peptidomiméticos/farmacologia , Peptidomiméticos/uso terapêutico , Inibidores de Serina Proteinase/farmacologiaRESUMO
The treatment of Alzheimer's disease and many other brain-related disorders is limited because of the presence of the blood-brain barrier, which highly regulate the crossing of drugs. Metal nanoparticles have unique features that could contribute to the development of new therapies for these diseases. Nanoparticles have the capacity to carry several molecules of a drug; furthermore, their unique physico-chemical properties allow, for example, photothermal therapy to produce molecular surgery to destroy tumor cells and toxic structures. Recently, we demonstrated that gold nanoparticles conjugated to the peptide CLPFFD are useful to destroy the toxic aggregates of ß-amyloid, similar to the ones found in the brains of patients with Alzheimer's disease. However, nanoparticles, like many other compounds, have null or very low capacity to cross the blood-brain barrier. In order to devise a strategy to improve drug delivery to the brain, here we introduced the peptide sequence THRPPMWSPVWP into the gold nanoparticle-CLPFFD conjugate. This peptide sequence interacts with the transferrin receptor present in the microvascular endothelial cells of the blood-brain barrier, thus causing an increase in the permeability of the conjugate in brain, as shown by experiments in vitro and in vivo. Our results are highly relevant for the therapeutic applications of gold nanoparticles for molecular surgery in the treatment of neurodegenerative diseases such as Alzheimer's disease.
Assuntos
Encéfalo/efeitos dos fármacos , Ouro/química , Nanopartículas Metálicas/química , Peptídeos/química , Receptores da Transferrina/química , Doença de Alzheimer/metabolismo , Animais , Barreira Hematoencefálica , Bovinos , Técnicas de Cocultura , Colorimetria/métodos , Células Endoteliais/citologia , Humanos , Masculino , Microcirculação , Nanotecnologia/métodos , Permeabilidade , Ratos , Ratos Sprague-Dawley , Soro/metabolismoRESUMO
Here we studied the capacity of N-MePhe-(N-MePhe)(3)-CONH(2), Cha-(N-MePhe)(3)-CONH(2), and 2Nal-(N-MePhe)(3)-CONH(2) to carry various drugs (cargos) in in vitro blood-brain barrier (BBB) models in order to determine the versatility of these peptides as BBB-shuttles for drug delivery to the brain. Using SPPS, the peptides were coupled to GABA, Nip, and ALA to examine their passive BBB permeation by means of PAMPA and their lipophilicity by IAMC. Unaided, these nonpermeating drugs alone did not cross the PAMPA barrier and the BBB passively; however, the peptides tested as potential BBB shuttles transferred them by passive transfer through the PAMPA phospholipid. The permeability of peptides that showed the highest permeability in PAMPA, and Ac-N-MePhe-(N-MePhe)(3)-CONH(2) as the parent peptide was also examined in bovine brain microvessel endothelial cells (BBMECs). These peptide-based BBB shuttles open up the possibility to overcome the formidable obstacle of the BBB, thereby achieving drug delivery to the brain.