RESUMO
N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.
Assuntos
Amidoidrolases/metabolismo , Encefalomielite Autoimune Experimental/enzimologia , Encefalomielite Autoimune Experimental/patologia , Esclerose Múltipla/enzimologia , Esclerose Múltipla/patologia , Amidoidrolases/genética , Animais , Progressão da Doença , Feminino , Lipopolissacarídeos , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/enzimologia , Neurônios Motores/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Oligodendroglia/metabolismo , Medula Espinal/enzimologiaRESUMO
Palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) are endogenous lipid mediators that suppress inflammation. Their actions are terminated by the intracellular cysteine amidase, N-acylethanolamine acid amidase (NAAA). Even though NAAA may offer a new target for anti-inflammatory therapy, the lipid-like structures and reactive warheads of current NAAA inhibitors limit the use of these agents as oral drugs. A series of novel benzothiazole-piperazine derivatives that inhibit NAAA in a potent and selective manner by a non-covalent mechanism are described. A prototype member of this class (8) displays high oral bioavailability, access to the central nervous system (CNS), and strong activity in a mouse model of multiple sclerosis (MS). This compound exemplifies a second generation of non-covalent NAAA inhibitors that may be useful in the treatment of MS and other chronic CNS disorders.
Assuntos
Amidoidrolases/antagonistas & inibidores , Modelos Animais de Doenças , Endocanabinoides/farmacologia , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Esclerose Múltipla/tratamento farmacológico , Ácidos Oleicos/farmacologia , Ácidos Palmíticos/farmacologia , Administração Oral , Amidas , Amidoidrolases/metabolismo , Animais , Relação Dose-Resposta a Droga , Endocanabinoides/administração & dosagem , Endocanabinoides/química , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Etanolaminas/administração & dosagem , Etanolaminas/química , Camundongos , Estrutura Molecular , Esclerose Múltipla/metabolismo , Ácidos Oleicos/administração & dosagem , Ácidos Oleicos/química , Ácidos Palmíticos/administração & dosagem , Ácidos Palmíticos/química , Relação Estrutura-AtividadeRESUMO
Our study was aimed at investigating whether complement, a complex of soluble and membrane-associated serum proteins, could, in addition to its well-documented post-synaptic activity, also pre-synaptically affect the release of classic neurotransmitters in central nervous system (CNS). Complement (dilution 1 : 10 to 1 : 10000) elicited the release of preloaded [(3) H]-d-aspartate ([(3) H]d-ASP) and endogenous glutamate from mouse cortical synaptosomes in a dilution-dependent manner. It also evoked [(3) H]d-ASP release from mouse hippocampal, cerebellar, and spinal cord synaptosomes, as well as from rat and human cortical nerve endings, but left unaltered the release of GABA, [(3) H]noradrenaline or [(3) H]acetylcholine. Lowering external Na(+) (from 140 to 40 mM) or Ca(2+) (from 1.2 to 0.1 mM) ions prevented the 1 : 300 complement-evoked [(3) H]d-ASP release from mouse cortical synaptosomes. Complement-induced releasing effect was unaltered in synaptosomes entrapped with the Ca(2+) ions chelator 1,2-bis-(2-aminophenoxy) ethane-N,N,N',N', tetra-acetic acid or with pertussis toxin. Nifedipine,/ω-conotoxin GVIA/ω-conotoxin MVIIC mixture as well as the vesicular ATPase blocker bafilomycin A1 were also inefficacious. The excitatory amino acid transporter blocker DL-threo-ß-benzyloxyaspartic acid, on the contrary, reduced the complement-evoked releasing effect in a concentration-dependent manner. We concluded that complement-induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier-mediated release. Our observations afford new insights into the molecular events accounting for immune and CNS crosstalk. We investigated whether complement, a complex of soluble and membrane-associated serum proteins, could pre-synaptically affect the release of classic neurotransmitters in the central nervous system (CNS). Our data provide evidence that complement-induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier-mediated release. Our observations add new insights to the knowledge of the molecular events accounting for immune and CNS crosstalk. EAAT = excitatory amino acid transporter.
Assuntos
Encéfalo/metabolismo , Proteínas do Sistema Complemento/metabolismo , Ácido Glutâmico/metabolismo , Medula Espinal/metabolismo , Sinaptossomos/metabolismo , Animais , Proteínas do Sistema Complemento/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Sinaptossomos/efeitos dos fármacosRESUMO
Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzymeand highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients. Glycogen synthase kinase 3ß (GSK-3ß) is a serine/threonine protein kinase mediating phosphorylation on serine and threonine amino acid residues of several target molecules. The enzyme is involved in the regulation of many cellular processes and aberrant activity of GSK-3ß has been linked to several disease conditions. There is now large evidence on the role of GSK-3ß in the pathophysiology of mood disturbances with special regard to bipolar disorders. In the present study we further investigated the role of GSK-3ß in bipolar disorders by studying AF3581, the prototype of a novel class of ATP-competitive GSK-3ß inhibitors having the common N-[(1- alkylpiperidin-4-yl) methyl]-1H-indazole-3-carboxamide scaffold. Based on previous studies, AF3581 inhibits GSK-3ß in the nanomolar range on purified human enzyme and highly selective with respect to other kinases. Current study demonstrates that the compound has efficacy both in the chronic mild stress paradigm of depression (mimicking the down phase of bipolar disorder) and on mice aggressiveness in the resident intruder model (mimicking the up phase). These findings underline the importance of aberrant GSK-3ß activity in the development/ maintenance of mood oscillation in this peculiar pathological condition. Moreover, the present work also suggests a therapeutic potential for selective GSK-3 ß inhibitors in the management of bipolar disorders patients.
Assuntos
Afeto/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Transtorno Bipolar/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Depressão/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Agressão/efeitos dos fármacos , Anedonia/efeitos dos fármacos , Animais , Transtorno Bipolar/enzimologia , Transtorno Bipolar/fisiopatologia , Transtorno Bipolar/psicologia , Encéfalo/enzimologia , Encéfalo/fisiopatologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Depressão/enzimologia , Depressão/fisiopatologia , Depressão/psicologia , Modelos Animais de Doenças , Preferências Alimentares/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Hidrocortisona/sangue , Locomoção/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , AutoimagemRESUMO
Novel treatments for bipolar disorder with improved efficacy and broader spectrum of activity are urgently needed. Glycogen synthase kinase 3ß (GSK-3ß) has been suggested to be a key player in the pathophysiology of bipolar disorder. A series of novel GSK-3ß inhibitors having the common N-[(1-alkylpiperidin-4-yl)methyl]-1H-indazole-3-carboxamide scaffold were prepared taking advantage of an X-ray cocrystal structure of compound 5 with GSK-3ß. We probed different substitutions at the indazole 5-position and at the piperidine-nitrogen to obtain potent ATP-competitive GSK-3ß inhibitors with good cell activity. Among the compounds assessed in the in vivo PK experiments, 14i showed, after i.p. dosing, encouraging plasma PK profile and brain exposure, as well as efficacy in a mouse model of mania. Compound 14i was selected for further in vitro/in vivo pharmacological evaluation, in order to elucidate the use of ATP-competitive GSK-3ß inhibitors as new tools in the development of new treatments for mood disorders.
Assuntos
Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Transtornos do Humor/tratamento farmacológico , Trifosfato de Adenosina/metabolismo , Anfetamina/farmacologia , Animais , Ligação Competitiva/efeitos dos fármacos , Células CHO , Estimulantes do Sistema Nervoso Central/farmacologia , Cricetinae , Cricetulus , Inibidores Enzimáticos/uso terapêutico , Ensaios de Triagem em Larga Escala , Humanos , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Atividade Motora/efeitos dos fármacos , Fosforilação , Relação Estrutura-Atividade , Difração de Raios X , Proteínas tau/metabolismoRESUMO
BACKGROUND AND PURPOSE: Altered glutamate exocytosis and cAMP production in cortical terminals of experimental autoimmune encephalomyelitis (EAE) mice occur at the early stage of disease (13 days post-immunization, d.p.i.). Neuronal defects were paralleled by overexpression of the central chemokine CCL5 (also known as RANTES), suggesting it has a role in presynaptic impairments. We propose that drugs able to restore CCL5 content to physiological levels could also restore presynaptic defects. Because of its efficacy in controlling CCL5 overexpression, desipramine (DMI) appeared to be a suitable candidate to test our hypothesis. EXPERIMENTAL APPROACH: Control and EAE mice at 13 d.p.i. were acutely or chronically administered DMI and monitored for behaviour and clinical scores. Noradrenaline and glutamate release, cAMP, CCL5 and TNF-α production were quantified in cortical synaptosomes and homogenates. Peripheral cytokine production was also determined. KEY RESULTS: Noradrenaline exocytosis and α2 -adrenoeceptor-mediated activity were unmodified in EAE mice at 13 d.p.i. when compared with control. Acute, but not chronic, DMI reduced CCL5 levels in cortical homogenates of EAE mice at 13 d.p.i., but did not affect peripheral IL-17 and TNF-α contents or CCL5 plasma levels. Acute DMI caused a long-lasting restoration of glutamate exocytosis, restored endogenous cAMP production and impeded the shift from inhibition to facilitation of the CCL5-mediated control of glutamate exocytosis. Finally, DMI ameliorated anxiety-related behaviour but not motor activity or severity of clinical signs. CONCLUSIONS: We propose DMI as an add-on therapy to normalize neuropsychiatric symptoms in multiple sclerosis patients at the early stage of the disease.