RESUMEN
Monoacylglycerol lipase (MGL) inhibition provides a potential treatment approach to glaucoma through the regulation of ocular 2-arachidonoylglycerol (2-AG) levels and the activation of CB1 receptors. Herein, we report the discovery of new series of carbamates as highly potent and selective MGL inhibitors. The new inhibitors showed potent nanomolar inhibitory activity against recombinant human and purified rat MGL, were selective (>1000-fold) against serine hydrolases FAAH and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Protein-based 1H NMR experiments indicated that inhibitor 2 rapidly formed a covalent adduct with MGL with a residence time of about 6â¯h. This interconversion process "intrinsic reversibility" was exploited by modifications of the ligand's size (length and bulkiness) to generate analogs with "tunable' adduct residence time (τ). Inhibitor 2 was evaluated in a normotensive murine model for assessing intraocular pressure (IOP), which could lead to glaucoma, a major cause of blindness. Inhibitor 2 was found to decrease ocular pressure by â¼4.5â¯mmHg in a sustained manner for at least 12â¯h after a single ocular application, underscoring the potential for topically-administered MGL inhibitors as a novel therapeutic target for the treatment of glaucoma.
Asunto(s)
Carbamatos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Glaucoma/tratamiento farmacológico , Monoacilglicerol Lipasas/antagonistas & inhibidores , Animales , Carbamatos/síntesis química , Carbamatos/química , Carbamatos/farmacocinética , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Humanos , Masculino , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Simulación del Acoplamiento Molecular , Estructura Molecular , Monoacilglicerol Lipasas/química , Piperazinas/síntesis química , Piperazinas/química , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Piperidinas/síntesis química , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/uso terapéutico , Ratas , Relación Estructura-ActividadRESUMEN
FAAH inhibitors offer safety advantages by augmenting the anandamide levels "on demand" to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed "precovalent" FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Fármacos Neuroprotectores/farmacología , Piperazina/farmacología , Piperidinas/farmacología , Amidohidrolasas/metabolismo , Animales , Diseño de Fármacos , Inhibidores Enzimáticos/química , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Fármacos Neuroprotectores/química , Piperazina/análogos & derivados , Piperidinas/química , RatasRESUMEN
An improved understanding of the endocannabinoid system has provided new avenues of drug discovery and development toward the management of pain and other behavioral maladies. Exogenous cannabinoid type 1 (CB1) receptor agonists such as Δ9-tetrahydrocannabinol are increasingly used for their medicinal actions; however, their utility is constrained by concern regarding abuse-related subjective effects. This has led to growing interest in the clinical benefit of indirectly enhancing the activity of the highly labile endocannabinoids N-arachidonoylethanolamine [AEA (or anandamide)] and/or 2-arachidonoylglycerol (2-AG) via catabolic enzyme inhibition. The present studies were conducted to determine whether such actions can lead to CB1 agonist-like subjective effects, as reflected in CB1-related discriminative stimulus effects in laboratory subjects. Squirrel monkeys (n = 8) that discriminated the CB1 full agonist AM4054 (0.01 mg/kg) from vehicle were used to study, first, the inhibitors of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MGL) alone or in combination [FAAH (URB597, AM4303); MGL (AM4301); FAAH/MGL (JZL195, AM4302)] and, second, the ability of the endocannabinoids AEA and 2-AG to produce CB1 agonist-like effects when administered alone or after enzyme inhibition. Results indicate that CB1-related discriminative stimulus effects were produced by combined, but not selective, inhibition of FAAH and MGL, and that these effects were nonsurmountably antagonized by low doses of rimonabant. Additionally, FAAH or MGL inhibition revealed CB1-like subjective effects produced by AEA but not by 2-AG. Taken together, the present data suggest that therapeutic effects of combined, but not selective, enhancement of AEA or 2-AG activity via enzyme inhibition may be accompanied by CB1 receptor-mediated subjective effects.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Aprendizaje Discriminativo/efectos de los fármacos , Drogas en Investigación/farmacología , Endocannabinoides/farmacología , Inhibidores Enzimáticos/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Receptor Cannabinoide CB1/agonistas , Adamantano/administración & dosificación , Adamantano/efectos adversos , Adamantano/análogos & derivados , Adamantano/farmacología , Amidohidrolasas/metabolismo , Animales , Ácidos Araquidónicos/administración & dosificación , Ácidos Araquidónicos/agonistas , Ácidos Araquidónicos/antagonistas & inhibidores , Ácidos Araquidónicos/farmacología , Conducta Animal/efectos de los fármacos , Antagonistas de Receptores de Cannabinoides/administración & dosificación , Antagonistas de Receptores de Cannabinoides/efectos adversos , Antagonistas de Receptores de Cannabinoides/farmacología , Cannabinol/administración & dosificación , Cannabinol/efectos adversos , Cannabinol/análogos & derivados , Cannabinol/farmacología , Relación Dosis-Respuesta a Droga , Agonismo de Drogas , Antagonismo de Drogas , Drogas en Investigación/administración & dosificación , Drogas en Investigación/efectos adversos , Endocannabinoides/administración & dosificación , Endocannabinoides/agonistas , Endocannabinoides/antagonistas & inhibidores , Inhibidores Enzimáticos/administración & dosificación , Inhibidores Enzimáticos/efectos adversos , Glicéridos/administración & dosificación , Glicéridos/agonistas , Glicéridos/antagonistas & inhibidores , Glicéridos/farmacología , Inyecciones Intramusculares , Inyecciones Intravenosas , Ligandos , Masculino , Monoacilglicerol Lipasas/metabolismo , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Alcamidas Poliinsaturadas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Receptor Cannabinoide CB1/metabolismo , SaimiriRESUMEN
The primary psychoactive ingredient of marijuana, Δ(9)-tetrahydrocannabinol (Δ(9)-THC), has medicinal value but also produces unwanted deleterious effects on cognitive function, promoting the search for improved cannabinergic therapeutics. The present studies used a battery of touchscreen procedures in squirrel monkeys to compare the effects of different types of cannabinergic drugs on several measures of performance including learning (repeated acquisition), cognitive flexibility (discrimination reversal), short-term memory (delayed matching-to-sample), attention (psychomotor vigilance), and motivation (progressive ratio). Drugs studied included the cannabinoid agonist Δ(9)-THC, fatty acid amide hydrolase (FAAH) inhibitor cyclohexylcarbamic acid 3-carbamoylbiphenyl-3-yl ester (URB597), and endocannabinoid anandamide and its stable synthetic analog methanandamide [(R)-(+)-arachidonyl-1'-hydroxy-2'-propylamide]. The effects of Δ(9)-THC and anandamide after treatment with the cannabinoid receptor type 1 inverse agonist/antagonist rimonabant [5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1Hpyrazole-3-carboxamide] and the FAAH inhibitor URB597, respectively, also were examined. The results showed the following: 1) Δ(9)-THC produced dose-related impairments of discrimination-based cognitive behavior with potency that varied across tasks (discriminative capability < learning < flexibility < short-term memory); 2) anandamide alone and URB597 alone were without effect on all endpoints; 3) anandamide following URB597 pretreatment and methanandamide had negligible effects on discriminative capability, learning, and reversal, but following large doses affected delayed matching-to-sample performance in some subjects; 4) all drugs, except anandamide and URB597, disrupted attention; and 5) progressive ratio breakpoints were generally unaffected by all drugs tested, suggesting little to no effect on motivation. Taken together, these data indicate that metabolically stable forms of anandamide may have lesser adverse effects on cognitive functions than Δ(9)-THC, possibly offering a therapeutic advantage in clinical settings.
Asunto(s)
Ácidos Araquidónicos/farmacología , Conducta Animal/efectos de los fármacos , Cognición/efectos de los fármacos , Dronabinol/farmacología , Endocannabinoides/farmacología , Alcamidas Poliinsaturadas/farmacología , Animales , Atención/efectos de los fármacos , Benzamidas/farmacología , Carbamatos/farmacología , Aprendizaje Discriminativo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Aprendizaje/efectos de los fármacos , Masculino , Memoria a Corto Plazo/efectos de los fármacos , Motivación/efectos de los fármacos , Piperidinas/farmacología , Desempeño Psicomotor/efectos de los fármacos , Pirazoles/farmacología , Aprendizaje Inverso/efectos de los fármacos , Rimonabant , SaimiriRESUMEN
Cannabinoid receptor 1 (CB(1)) inverse agonists (e.g., rimonabant) have been reported to produce adverse effects including nausea, emesis, and anhedonia that limit their clinical applications. Recent laboratory studies suggest that the effects of CB(1) neutral antagonists differ from those of such inverse agonists, raising the possibility of improved clinical utility. However, little is known regarding the antagonist properties of neutral antagonists. In the present studies, the CB(1) inverse agonist SR141716A (rimonabant) and the CB(1) neutral antagonist AM4113 were compared for their ability to modify CB(1) receptor-mediated discriminative stimulus effects in nonhuman primates trained to discriminate the novel CB(1) full agonist AM4054. Results indicate that AM4054 serves as an effective CB(1) discriminative stimulus, with an onset and time course of action comparable with that of the CB(1) agonist Δ(9)-tetrahydrocannabinol, and that the inverse agonist rimonabant and the neutral antagonist AM4113 produce dose-related rightward shifts in the AM4054 dose-effect curve, indicating that both drugs surmountably antagonize the discriminative stimulus effects of AM4054. Schild analyses further show that rimonabant and AM4113 produce highly similar antagonist effects, as evident in comparable pA(2) values (6.9). Taken together with previous studies, the present data suggest that the improved safety profile suggested for CB(1) neutral antagonists over inverse agonists is not accompanied by a loss of antagonist action at CB(1) receptors.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Antagonistas de Receptores de Cannabinoides/farmacología , Cannabinoides/farmacología , Discriminación en Psicología/efectos de los fármacos , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Relación Dosis-Respuesta a Droga , Dronabinol/farmacología , Ligandos , Masculino , Piperidinas/farmacología , Pirazoles/farmacología , Rimonabant , SaimiriRESUMEN
Δ-Tetrahydrocannabinol (THC) has been characterized as a partial agonist at cannabinoid CB1 receptors in vitro; however, it often produces the same maximum effects in vivo as other cannabinoid agonists. This study was carried out to determine whether THC would antagonize the hypothermic effects of another cannabinoid agonist, AM2389, in mice. Male mice were injected with 1-100 mg/kg THC, 0.01-0.1 mg/kg AM2389, or a combination of 30 mg/kg THC and 0.1-1.0 mg/kg AM2389, and rectal temperature was recorded for up to 12 h after injection. THC reduced the temperature by 5.6°C at a dose of 30 mg/kg; further increases in the dose did not produce larger effects, indicating a plateau in the THC dose-effect function. AM2389 reduced temperature by 9.0°C at a dose of 0.1 mg/kg. One hour pretreatment with 30 mg/kg THC attenuated the hypothermic effects of 0.1 mg/kg AM2389; a 10-fold higher dose, 1.0 mg/kg AM2389, was required to further decrease temperature, reflecting a five-fold rightward shift of the lower portion of the AM2389 dose-effect function following THC pretreatment. These results indicate that, in an assay of mouse hypothermia, THC exerts both agonist and antagonist effects following acute administration, and mark the first demonstration of partial agonist/antagonist effects of THC in vivo.
Asunto(s)
Agonistas de Receptores de Cannabinoides/farmacología , Dronabinol/farmacología , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB1/antagonistas & inhibidores , Animales , Benzopiranos/administración & dosificación , Benzopiranos/farmacología , Agonistas de Receptores de Cannabinoides/administración & dosificación , Relación Dosis-Respuesta a Droga , Dronabinol/administración & dosificación , Hipotermia/inducido químicamente , Hipotermia/prevención & control , Masculino , Ratones , Factores de TiempoRESUMEN
A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with K(i) values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.
Asunto(s)
Benzopiranos/química , Cannabinoides/química , Ligandos , Receptor Cannabinoide CB2/química , Tiofenos/química , Benzopiranos/síntesis química , Sitios de Unión , Cannabinoides/metabolismo , Células HEK293 , Humanos , Unión Proteica , Receptor Cannabinoide CB1/química , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Rayos UltravioletaRESUMEN
We describe the use of a microwave reaction for the conversion of various bromides to sodium sulfonates that have been further elaborated to sulfonyl chlorides. This new approach leads to much improved yields and shorter reaction times. Representative sulfonyl chlorides serve as precursors for the respective sulfonyl fluorides that are potent inhibitors of the fatty acid amide hydrolase.
RESUMEN
The anticholinesterase paraoxon (Pxn) is related to military nerve agents that increase acetylcholine levels, trigger seizures, and cause excitotoxic damage in the brain. In rat hippocampal slice cultures, high-dose Pxn was applied resulting in a presynaptic vulnerability evidenced by a 64% reduction in synapsin IIb (syn IIb) levels, whereas the postsynaptic protein GluR1 was unchanged. Other signs of Pxn-induced cytotoxicity include the oxidative stress-related production of stable 4-hydroxynonenal (4-HNE)-protein adducts. Next, the Pxn toxicity was tested for protective effects by the fatty acid amide hydrolase (FAAH) inhibitor AM5206, a compound linked to enhanced repair signaling through the endocannabinoid pathway. The Pxn-mediated declines in syn IIb and synaptophysin were prevented by AM5206 in the slice cultures. To test if the protective results in the slice model translate to an in vivo model, AM5206 was injected i.p. into rats, followed immediately by subcutaneous Pxn administration. The toxin caused a pathogenic cascade initiated by seizure events, leading to presynaptic marker decline and oxidative changes in the hippocampus and frontal cortex. AM5206 exhibited protective effects including the reduction of seizure severity by 86%, and improving balance and coordination measured 24 h post-insult. As observed in hippocampal slices, the FAAH inhibitor also prevented the Pxn-induced loss of syn IIb in vivo. In addition, the AM5206 compound reduced the 4-HNE modifications of proteins and the ß1 integrin activation events both in vitro and in vivo. These results indicate that Pxn exposure produces oxidative and synaptic toxicity that leads to the behavioral deficits manifested by the neurotoxin. In contrast, the presence of FAAH inhibitor AM5206 offsets the pathogenic cascade elicited by the Pxn anticholinesterase.
Asunto(s)
Endocannabinoides/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Fármacos Neuroprotectores/uso terapéutico , Paraoxon/toxicidad , Éteres Fenílicos/uso terapéutico , Convulsiones/tratamiento farmacológico , Amidohidrolasas/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/farmacología , Lóbulo Frontal/efectos de los fármacos , Lóbulo Frontal/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Insecticidas/toxicidad , Masculino , Fármacos Neuroprotectores/farmacología , Éteres Fenílicos/farmacología , Ratas , Ratas Sprague-Dawley , Convulsiones/etiología , Sinaptofisina/metabolismoRESUMEN
RATIONALE: Previous reports shows rimonabant's inverse properties may be a limiting factor for treating cannabinoid dependence. To overcome this limitation, neutral antagonists were developed, to address mechanisms by which an inverse agonist and neutral antagonist elicit withdrawal. OBJECTIVE: The objective of this study is to introduce an animal model to study cannabinoid dependence by incorporating traditional methodologies and profiling novel cannabinoid ligands with distinct pharmacological properties/modes of action by evaluating their pharmacological effects on CB1-receptor (CB1R) related physiological/behavioral endpoints. METHODS: The cannabinergic AM2389 was acutely characterized in the tetrad (locomotor activity, analgesia, inverted screen/catalepsy bar test, and temperature), with some comparisons made to Δ(9)-tetrahydrocannabinol (THC). Tolerance was measured in mice repeatedly administered AM2389. Antagonist-precipitated withdrawal was characterized in cannabinoid-adapted mice induced by either centrally acting antagonists, rimonabant and AM4113, or an antagonist with limited brain penetration, AM6545. RESULTS: In the tetrad, AM2389 was more potent and longer acting than THC, suggesting a novel approach for inducing dependence. Repeated administration of AM2389 led to tolerance by attenuating hypothermia that was induced by acute AM2389 administration. Antagonist-precipitated withdrawal signs were induced by rimonabant or AM4113, but not by AM6545. Antagonist-precipitated withdrawal was reversed by reinstating AM2389 or THC. CONCLUSIONS: These findings suggest cannabinoid-precipitated withdrawal may not be ascribed to the inverse properties of rimonabant, but rather to rapid competition with the agonist at the CB1R. This withdrawal syndrome is likely centrally mediated, since only the centrally acting CB1R antagonists elicited withdrawal, i.e., such responses were absent after the purported peripherally selective CB1R antagonist AM6545.
Asunto(s)
Cannabinoides/efectos adversos , Dronabinol/farmacología , Morfolinas/farmacología , Piperidinas/farmacología , Pirazoles/farmacología , Síndrome de Abstinencia a Sustancias , Animales , Temperatura Corporal/efectos de los fármacos , Antagonistas de Receptores de Cannabinoides/farmacología , Modelos Animales de Enfermedad , Tolerancia a Medicamentos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Receptor Cannabinoide CB1/antagonistas & inhibidores , RimonabantRESUMEN
The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptor's ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677's ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677's topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.
Asunto(s)
Ácidos Araquidónicos/farmacología , Agonistas de Receptores de Cannabinoides/farmacología , Isotiocianatos/farmacología , Animales , Ácidos Araquidónicos/química , Agonistas de Receptores de Cannabinoides/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colforsina , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Endocitosis/efectos de los fármacos , Células HEK293 , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Enlace de Hidrógeno , Isotiocianatos/química , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Mutación , Ensayo de Unión Radioligante , Receptor Cannabinoide CB1/genética , Receptor Cannabinoide CB1/metabolismo , TransfecciónRESUMEN
RATIONALE: The endocannabinoid signaling system (ECS) has been targeted for developing novel therapeutics since ECS dysfunction has been implicated in various pathologies. Current focus is on chemical modifications of the hexahydrocannabinol (HHC) nabilone (Cesamet(®)). OBJECTIVE: To characterize the novel, high-affinity cannabinoid receptor 1 (CB(1)R) HHC-ligand AM2389 [9ß-hydroxy-3-(1-hexyl-cyclobut-1-yl)-hexahydrocannabinol in two rodent pre-clinical assays. MATERIALS AND METHODS: CB(1)R mediation of AM2389-induced hypothermia in mice was evaluated with AM251, a CB(1)R-selective antagonist/inverse agonist. Additionally, two groups of rats discriminated the full cannabinergic aminoalkylindole AM5983 (0.18 and 0.56 mg/kg) from vehicle 20 min post-injection in a two-choice operant conditioning task motivated by 0.1% saccharin/water. Generalization/substitution tests were conducted with AM2389, AM5983, and Δ(9)-tetrahydrocannabinol (Δ(9)-THC). RESULTS: Δ(9)-THC (30 mg/kg)-induced hypothermia exhibited a faster onset and shorter duration of action compared with AM2389 (0.1 and 0.3 mg/kg). AM251 (3 and 10 mg/kg) attenuated/blocked hypothermia induced by 0.3 mg/kg AM2389. In drug discrimination, the order of potency was AM2389 > AM5983 > Δ(9)-THC with ED(50) values of 0.0025, 0.0571, and 0.2635 mg/kg, respectively, in the low-dose condition. The corresponding ED(50) values in the high-dose condition were 0.0069, 0.1246, and 0.8438 mg/kg, respectively. Onset of the effects of AM2389 was slow with a protracted time-course; the functional, perceptual in vivo half-life was approximately 17 h. CONCLUSIONS: This potent cannabinergic HHC exhibited a slow onset of action with a protracted time-course. The AM2389 chemotype appears well suited for further drug development, and AM2389 currently is used to probe behavioral consequences of sustained ECS activation.
Asunto(s)
Benzopiranos/farmacología , Hipotermia/inducido químicamente , Receptor Cannabinoide CB1/agonistas , Animales , Benzopiranos/antagonistas & inhibidores , Condicionamiento Operante/efectos de los fármacos , Discriminación en Psicología/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Dronabinol/farmacología , Interacciones Farmacológicas , Indoles/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/antagonistas & inhibidores , Factores de TiempoRESUMEN
In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu(169);Leu(176))-to-Ser(Ser(169);Ser(176)) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser(122) with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wild-type hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser(122) residue as compared to that of Cys(122) in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys(122) residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser(122), and the mechanisms underlying hMGL inhibition by different classes of small molecules.
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Endocannabinoides/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/metabolismo , Ingeniería de Proteínas/métodos , Compuestos de Sulfhidrilo/metabolismo , Sustitución de Aminoácidos/genética , Endocannabinoides/química , Endocannabinoides/genética , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Monoacilglicerol Lipasas/genética , Mutación , Solubilidad , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/farmacologíaRESUMEN
Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.
Asunto(s)
Alcanosulfonatos/farmacología , Amidohidrolasas/antagonistas & inhibidores , Encéfalo/efectos de los fármacos , Palmitatos/farmacología , Fenoles/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Amidohidrolasas/metabolismo , Animales , Encéfalo/metabolismo , Humanos , Masculino , Ratones , Modelos Moleculares , Estructura Molecular , Ensayo de Unión Radioligante , Ratas , Proteínas Recombinantes/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-ActividadRESUMEN
Advances in the understanding of the endogenous cannabinoid system have led to several therapeutic indications for new classes of compounds that enhance cannabinergic responses. Endocannabinoid levels are elevated during pathogenic conditions, and inhibitors of endocannabinoid inactivation promote such on-demand responses. The endocannabinoids anandamide and 2-arachidonoyl glycerol have been implicated in protective signaling against excitotoxic episodes, including seizures. To better understand modulatory pathways that can exploit such responses, we used the new generation compound AM6701 that blocks both the anandamide-deactivating enzyme fatty acid amide hydrolase (FAAH) and the 2-arachidonoyl glycerol-deactivating enzyme monoacylglycerol lipase (MAGL) with equal potency. Also studied was the structural isomer AM6702 which is 44-fold more potent for inhibiting FAAH versus MAGL. When applied before and during kainic acid (KA) exposure to cultured hippocampal slices, AM6701 protected against the resulting excitotoxic events of calpain-mediated cytoskeletal damage, loss of presynaptic and postsynaptic proteins, and pyknotic changes in neurons. The equipotent inhibitor was more effective than its close relative AM6702 at protecting against the neurodegenerative cascade assessed in the slice model. In vivo, AM6701 was also the more effective compound for reducing the severity of KA-induced seizures and protecting against behavioral deficits linked to seizure damage. Corresponding with the behavioral improvements, cytoskeletal and synaptic protection was elicited by AM6701, as found in the KA-treated hippocampal slice model. It is proposed that the influence of AM6701 on FAAH and MAGL exerts a synergistic action on the endocannabinoid system, thereby promoting the protective nature of cannabinergic signaling to offset excitotoxic brain injury.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Endocannabinoides/metabolismo , Monoacilglicerol Lipasas/antagonistas & inhibidores , Neuronas/enzimología , Convulsiones/enzimología , Animales , Convulsivantes/toxicidad , Inhibidores Enzimáticos/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Immunoblotting , Masculino , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Tetrazoles/toxicidadRESUMEN
In pursuit of a more detailed understanding of the structural requirements for the key side chain cannabinoid pharmacophore, we have extended our SAR to cover a variety of conformationally modified side chains within the 9-keto and 9-hydroxyl tricyclic structures. Of the compounds described here, those with a seven-atom long side chain substituted with a cyclopentyl ring at C1' position have very high affinities for both CB1 and CB2 (0.97 nM < K(i) < 5.25 nM), with no preference for either of the two receptors. However, presence of the smaller cyclobutyl group at the C1' position leads to an optimal affinity and selectivity interaction with CB1. Thus, two of the C1'-cyclobutyl analogues, namely, (6aR,10aR)-3-(1-hexyl-cyclobut-1-yl)-6,6a,7,8,10,10a-hexahydro-1-hydroxy-6,6-dimethyl-9H-dibenzo[b,d]pyran-9-one and (6aR,9R,10aR)-3-(1-hexyl-cyclobut-1-yl)-6a,7,8,9,10,10a-hexahydro-6,6-dimethyl-6H-dibenzo[b,d]pyran-1,9 diol (7e-ß, AM2389), exhibited remarkably high affinities (0.84 and 0.16 nM, respectively) and significant selectivities (16- and 26-fold, respectively) for CB1. Compound 7e-ß was found to exhibit exceptionally high in vitro and in vivo potency with a relatively long duration of action.
Asunto(s)
Analgésicos/síntesis química , Benzopiranos/síntesis química , Cannabinol/análogos & derivados , Cannabinol/síntesis química , Receptor Cannabinoide CB1/agonistas , Analgesia , Analgésicos/química , Analgésicos/farmacología , Animales , Benzopiranos/química , Benzopiranos/farmacología , Cannabinol/química , Cannabinol/farmacología , Línea Celular , Femenino , Hipotermia Inducida , Técnicas In Vitro , Ratones , Modelos Moleculares , Prosencéfalo/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB2/agonistas , Bazo/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Sinaptosomas/metabolismoRESUMEN
A mild, selective, and high-yielding method for oxidation of sulfides to sulfoxides using IBX and tetraethylammonium bromide in a variety of solvents is described. The method offers the advantage of short reaction times, no over-oxidation to sulfones, and compatibility to a wide range of functional groups.