RESUMO
Monoacylglycerol lipase (MGL) is a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Previous efforts to design MGL inhibitors have focused on chemical scaffolds that irreversibly block the activity of this enzyme. Here, we describe two naturally occurring terpenoids, pristimerin and euphol, which inhibit MGL activity with high potency (median effective concentration, IC(50) = 93 nM and 315 nM, respectively) through a reversible mechanism. Mutational and modeling studies suggest that the two agents occupy a common hydrophobic pocket located within the putative lid domain of MGL, and each reversibly interacts with one of two adjacent cysteine residues (Cys(201) and Cys(208)) flanking such pocket. This previously unrecognized regulatory region might offer a molecular target for potent and reversible inhibitors of MGL.
Assuntos
Inibidores Enzimáticos/farmacologia , Lanosterol/análogos & derivados , Monoacilglicerol Lipases/antagonistas & inibidores , Triterpenos/farmacologia , Animais , Ácidos Araquidônicos/metabolismo , Sítios de Ligação , Células Cultivadas , Desenho de Fármacos , Inibidores Enzimáticos/química , Glicerol/análogos & derivados , Glicerol/metabolismo , Células HeLa , Humanos , Lanosterol/química , Lanosterol/farmacologia , Simulação de Dinâmica Molecular , Monoacilglicerol Lipases/metabolismo , Neurônios/efeitos dos fármacos , Triterpenos Pentacíclicos , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Triterpenos/químicaRESUMO
The fatty acid ethanolamides are a class of signaling lipids that include agonists at cannabinoid and alpha type peroxisome proliferator-activated receptors (PPARalpha). In the brain, these compounds are primarily hydrolyzed by the intracellular serine enzyme fatty acid amide hydrolase (FAAH). O-aryl carbamate FAAH inhibitors such as URB597 are being evaluated clinically for the treatment of pain and anxiety, but interactions with carboxylesterases in liver might limit their usefulness. Here we explore two strategies aimed at overcoming this limitation. Lipophilic N-terminal substitutions, which enhance FAAH recognition, yield potent inhibitors but render such compounds susceptible to attack by broad-spectrum hydrolases and inactive in vivo. By contrast, polar electron-donating O-aryl substituents, which decrease carbamate reactivity, yield compounds, such as URB694, that are highly potent FAAH inhibitors in vivo and less reactive with off-target carboxylesterases. The results suggest that an approach balancing inhibitor reactivity with target recognition produces FAAH inhibitors that display significantly improved drug-likeness.
Assuntos
Amidoidrolases/antagonistas & inibidores , Carbamatos/química , Inibidores Enzimáticos/química , Amidoidrolases/metabolismo , Animais , Compostos de Bifenilo/química , Compostos de Bifenilo/farmacocinética , Carbamatos/farmacocinética , Carbamatos/farmacologia , Carboxilesterase/metabolismo , Inibidores Enzimáticos/farmacologia , Meia-Vida , Masculino , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
BACKGROUND: CB(1) cannabinoid receptors in the brain are known to participate in the regulation of reward-based behaviors. However, the contribution of each of the endocannabinoid transmitters, anandamide and 2-arachidonoylglycerol (2-AG), to these behaviors remains undefined. To address this question, we assessed the effects of URB597, a selective anandamide deactivation inhibitor, as a reinforcer of drug-seeking and drug-taking behavior in squirrel monkeys. METHODS: We investigated the reinforcing effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 in monkeys trained to intravenously self-administer Delta(9)-tetrahydrocannabinol (THC), anandamide, or cocaine and quantified brain endocannabinoid levels using liquid chromatography/mass spectrometry. We measured brain FAAH activity using an ex vivo enzyme assay. RESULTS: URB597 (.3 mg/kg, intravenous) blocked FAAH activity and increased anandamide levels throughout the monkey brain. This effect was accompanied by a marked compensatory decrease in 2-AG levels. Monkeys did not self-administer URB597, and the drug did not promote reinstatement of extinguished drug-seeking behavior previously maintained by THC, anandamide, or cocaine. Pretreatment with URB597 did not modify self-administration of THC or cocaine, even though, as expected, it significantly potentiated anandamide self-administration. CONCLUSIONS: In the monkey brain, the FAAH inhibitor URB597 increases anandamide levels while causing a compensatory down-regulation in 2-AG levels. These effects are accompanied by a striking lack of reinforcing properties, which distinguishes URB597 from direct-acting cannabinoid agonists such as THC. Our results reveal an unexpected functional heterogeneity within the endocannabinoid signaling system and suggest that FAAH inhibitors might be used therapeutically without risk of abuse or triggering of relapse to drug abuse.
Assuntos
Amidoidrolases/metabolismo , Ácidos Araquidônicos/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Dronabinol/administração & dosagem , Alcamidas Poli-Insaturadas/administração & dosagem , Reforço Psicológico , Análise de Variância , Animais , Comportamento Animal/efeitos dos fármacos , Benzamidas/farmacologia , Encéfalo/anatomia & histologia , Moduladores de Receptores de Canabinoides/metabolismo , Carbamatos/farmacologia , Cromatografia Líquida/métodos , Cocaína/administração & dosagem , Relação Dose-Resposta a Droga , Endocanabinoides , Masculino , Ratos , Ratos Wistar , Esquema de Reforço , Saimiri , Autoadministração/métodosRESUMO
Alkylcarbamic acid biphenyl-3-yl esters are a class of fatty acid amide hydrolase (FAAH) inhibitors that comprises cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester (URB597), a compound with analgesic, anxiolytic-like and antidepressant-like properties in rat and mouse models. Here, we extended the structure-activity relationships (SARs) for this class of compounds by replacing the cyclohexyl ring of the parent compound cyclohexylcarbamic acid biphenyl-3-yl ester (URB524) (FAAH IC50 = 63 nM) with a selected set of substituents of different size, shape, flexibility, and lipophilicity. Docking experiments and linear interaction energy (LIE) calculations indicated that the N-terminal group of O-arylcarbamates fits within the lipophilic region of the substrate-binding site, mimicking the arachidonoyl chain of anandamide. Significant potency improvements were observed for the beta-naphthylmethyl derivative 4q (IC50 = 5.3 nM) and its 3'-carbamoylbiphenyl-3-yl ester 4z (URB880, IC50 = 0.63 nM), indicating that shape complementarity and hydrogen bonds are crucial to obtain highly potent inhibitors.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Compostos de Bifenilo/síntese química , Carbamatos/síntese química , Relação Quantitativa Estrutura-Atividade , Compostos de Bifenilo/química , Carbamatos/química , Ligação de Hidrogênio , Modelos MolecularesRESUMO
The N-aryl carbamate URB602 (biphenyl-3-ylcarbamic acid cyclohexyl ester) is an inhibitor of monoacylglycerol lipase (MGL), a serine hydrolase involved in the biological deactivation of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG). Here, we investigated the mechanism by which URB602 inhibits purified recombinant rat MGL by using a combination of biochemical and structure-activity relationship (SAR) approaches. We found that URB602 weakly inhibits recombinant MGL (IC(50) = 223 +/- 63 microM) through a rapid and noncompetitive mechanism. Dialysis experiments and SAR analyses suggest that URB602 acts through a partially reversible mechanism rather than by irreversible carbamoylation of MGL. Finally, URB602 (100 microM) elevates 2-AG levels in hippocampal slice cultures without affecting levels of other endocannabinoid-related substances. Thus, URB602 may provide a useful tool by which to investigate the physiological roles of 2-AG and explore the potential interest of MGL as a therapeutic target.
Assuntos
Ácidos Araquidônicos/metabolismo , Compostos de Bifenilo/farmacologia , Encéfalo/efeitos dos fármacos , Glicerídeos/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Amidas , Animais , Ácidos Araquidônicos/química , Ácidos Araquidônicos/farmacologia , Compostos de Bifenilo/química , Encéfalo/metabolismo , Catálise/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Endocanabinoides , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Etanolaminas , Células HeLa , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Humanos , Cinética , Masculino , Monoacilglicerol Lipases/genética , Monoacilglicerol Lipases/metabolismo , Organofosfonatos/química , Organofosfonatos/farmacologia , Ácidos Palmíticos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Acute administration of neuropeptide Y (NPY) modulates alcohol intake in genetic and chemical models of high intake, while leaving intake unaffected during 'normal' or baseline conditions. In non-selected, normal rat lines, alcohol consumption can be increased by prolonged exposure to alcohol, and it is unclear what effect a constitutive increase in NPY function will have on alcohol intake. The purpose of the present study was to examine the effects on alcohol intake of an inducible, constitutive overexpression of NPY, one of the most abundant neuropeptides in the central nervous system. A liquid diet was used in combination with repeated alcohol deprivation sessions to increase alcohol intake in normal Wistar rats. We then examined the effect of NPY overexpression in the amygdala on excessive alcohol intake produced by prolonged exposure to alcohol and alcohol deprivation. Repeated withdrawal increased alcohol consumption in a 24-h continuous access two-bottle choice model. Both the number of withdrawals as well as the length of the withdrawal periods affected alcohol consumption with an increased intake resulting from multiple withdrawals and the alcohol deprivation effect being enhanced by longer periods of abstinence. The increase in intake following repeated abstinence was blunted by intra-amygdala administration of a Sindbis viral vector containing NPY cDNA. Amygdala NPY overexpression also was demonstrated to be anxiolytic in the open field test. Repeated withdrawal in combination with a history of alcohol consumption significantly elevated alcohol intake, and the amygdala may mediate the transition to high-drinking states in this model.
Assuntos
Alcoolismo/etiologia , Tonsila do Cerebelo/metabolismo , Ansiolíticos/metabolismo , Neuropeptídeo Y/metabolismo , Consumo de Bebidas Alcoólicas , Alcoolismo/metabolismo , Tonsila do Cerebelo/química , Animais , Peso Corporal , Dieta , Ingestão de Líquidos , Expressão Gênica , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Masculino , Modelos Animais , Neuropeptídeo Y/análise , Neuropeptídeo Y/genética , Ratos , Ratos Wistar , Sindbis virus/genética , Transdução Genética/métodosRESUMO
The application of transcriptomics and proteomics approaches to accurately dissected anatomically-defined brain regions and sub-regions remains a central focus of current neurobiological investigations as well as a necessary step towards single-neuron neurogenomics and neuroproteomics. A protocol is described for the simple, rapid, and reproducible laser microdissection of brain regions and sub-regions for microarray-based gene expression analyses from individual rats or mice using two rounds of in vitro transcription (IVT). The results presented also demonstrate that the current Affymetrix GeneChip arrays are well suited for this experimental design with high reproducibility and limited effects of the shortening of target RNA caused by the double IVT approach.
Assuntos
Encéfalo/fisiologia , Perfilação da Expressão Gênica/métodos , Lasers , Microdissecção/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Animais , Microdissecção/instrumentação , RatosRESUMO
Mammalian target of rapamycin (mTOR) is a key regulator of translational capacity. The mTOR inhibitor rapamycin can prevent forms of protein synthesis-dependent synaptic plasticity such as long-term facilitation in Aplysia and late-phase long-term potentiation (L-LTP) in the hippocampal CA1 region of rodents. In the latter model, two issues remain to be addressed: defining the L-LTP phase sensitive to rapamycin and identifying the site of rapamycin-sensitive protein synthesis. Here, we show that L-LTP is sensitive to application of rapamycin only during the induction paradigm, whereas rapamycin application after the establishment of L-LTP was ineffective. Second, we observed that Thr-389-phosphorylated p70 S6 kinase (p70S6K), the main active phosphoform of the mTOR effector p70S6K, was induced in an N-methyl-D-aspartate and phosphatidylinositol 3-kinase-dependent manner throughout the dendrites but not in the cell bodies of CA1 neurons in hippocampal slices after L-LTP induction. A similar dendrite-wide activation of p70S6K was induced in primary hippocampal neurons by depolarization with KCL or glutamate. In primary hippocampal neurons, the sites of dendritic activation of p70S6K appeared as discrete compartments along dendritic shafts like the hotspots for fast dendritic translation. Conversely, only a subset of dendritic spines also displayed activated p70S6K. Taken together, the present data suggest that the N-methyl-d-aspartate-, phosphatidylinositol 3-kinase-dependent dendritic activation of the mTOR-p70S6K pathway is necessary for the induction phase of protein synthesis-dependent synaptic plasticity. Newly synthesized proteins in dendritic shafts could be targeted selectively to activity-tagged synapses. Thus, coordinated activation of dendrite-wide translation and synaptic-specific activation is likely to be necessary for long-term synaptic plasticity.