RESUMEN
Monoacylglycerol lipase (MGLL) is the primary degradative enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). The first MGLL inhibitors have recently entered clinical development for the treatment of neurologic disorders. To support this clinical path, we report the pharmacological characterization of the highly potent and selective MGLL inhibitor ABD-1970 [1,1,1,3,3,3-hexafluoropropan-2-yl 4-(2-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-4-chlorobenzyl)piperazine-1-carboxylate]. We used ABD-1970 to confirm the role of MGLL in human systems and to define the relationship between MGLL target engagement, brain 2-AG concentrations, and efficacy. Because MGLL contributes to arachidonic acid metabolism in a subset of rodent tissues, we further used ABD-1970 to evaluate whether selective MGLL inhibition would affect prostanoid production in several human assays known to be sensitive to cyclooxygenase inhibitors. ABD-1970 robustly elevated brain 2-AG content and displayed antinociceptive and antipruritic activity in a battery of rodent models (ED50 values of 1-2 mg/kg). The antinociceptive effects of ABD-1970 were potentiated when combined with analgesic standards of care and occurred without overt cannabimimetic effects. ABD-1970 also blocked 2-AG hydrolysis in human brain tissue and elevated 2-AG content in human blood without affecting stimulated prostanoid production. These findings support the clinical development of MGLL inhibitors as a differentiated mechanism to treat pain and other neurologic disorders.
Asunto(s)
Endocannabinoides/metabolismo , Inhibidores Enzimáticos/farmacología , Monoacilglicerol Lipasas/antagonistas & inhibidores , Analgésicos/farmacología , Animales , Antipruriginosos/farmacología , Ácidos Araquidónicos/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular Tumoral , Inhibidores de la Ciclooxigenasa/farmacología , Glicéridos/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos ICR , Células PC-3 , Dolor/tratamiento farmacológico , Dolor/metabolismo , Piperidinas/farmacología , Prostaglandinas/farmacología , Ratas , Ratas Sprague-Dawley , RoedoresRESUMEN
The serine hydrolase monoacylglycerol lipase (MGLL) converts the endogenous cannabinoid receptor agonist 2-arachidonoylglycerol (2-AG) and other monoacylglycerols into fatty acids and glycerol. Genetic or pharmacological inactivation of MGLL leads to elevation in 2-AG in the central nervous system and corresponding reductions in arachidonic acid and eicosanoids, producing antinociceptive, anxiolytic, and antineuroinflammatory effects without inducing the full spectrum of psychoactive effects of direct cannabinoid receptor agonists. Here, we report the optimization of hexafluoroisopropyl carbamate-based irreversible inhibitors of MGLL, culminating in a highly potent, selective, and orally available, CNS-penetrant MGLL inhibitor, 28 (ABX-1431). Activity-based protein profiling experiments verify the exquisite selectivity of 28 for MGLL versus other members of the serine hydrolase class. In vivo, 28 inhibits MGLL activity in rodent brain (ED50 = 0.5-1.4 mg/kg), increases brain 2-AG concentrations, and suppresses pain behavior in the rat formalin pain model. ABX-1431 (28) is currently under evaluation in human clinical trials.
Asunto(s)
Descubrimiento de Drogas , Monoacilglicerol Lipasas/antagonistas & inhibidores , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/enzimología , Piperazina/farmacología , Piperazinas/farmacología , Pirrolidinas/farmacología , Animales , Perros , Relación Dosis-Respuesta a Droga , Humanos , Masculino , Ratones , Terapia Molecular Dirigida , Dolor/tratamiento farmacológico , Dolor/enzimología , Piperazina/farmacocinética , Piperazina/uso terapéutico , Piperazinas/farmacocinética , Piperazinas/uso terapéutico , Pirrolidinas/farmacocinética , Pirrolidinas/uso terapéutico , Ratas , Distribución TisularRESUMEN
We report herein the identification of MK-4409, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor. Starting from a high throughput screening (HTS) hit, medicinal chemistry efforts focused on optimizing of FAAH inhibition in vitro potency, improving the pharmacokinetic (PK) profile, and increasing in vivo efficacy in rodent inflammatory and neuropathic pain assays.
RESUMEN
Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.
Asunto(s)
Alérgenos/administración & dosificación , Asma/prevención & control , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Ascaris suum/inmunología , Asma/etiología , Asma/fisiopatología , Broncoconstricción/efectos de los fármacos , Broncoconstricción/inmunología , Broncoconstricción/fisiología , Degranulación de la Célula/efectos de los fármacos , Modelos Animales de Enfermedad , Humanos , Péptidos y Proteínas de Señalización Intracelular/fisiología , Macaca fascicularis , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Ovalbúmina/inmunología , Proteínas Tirosina Quinasas/fisiología , Ratas , Ratas Endogámicas BN , Ratas Sprague-Dawley , Ovinos , Transducción de Señal/efectos de los fármacos , Quinasa SykRESUMEN
The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Glicéridos/metabolismo , Monoacilglicerol Lipasas/metabolismo , Receptor Cannabinoide CB1/metabolismo , Receptor Cannabinoide CB2/metabolismo , Transducción de Señal/fisiología , Animales , Células COS , Línea Celular , Movimiento Celular , Chlorocebus aethiops , Endocannabinoides , Potenciales Postsinápticos Excitadores/fisiología , Técnicas de Silenciamiento del Gen , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Microscopía Electrónica de Transmisión , Neuronas/metabolismo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , TransfecciónRESUMEN
Gamma-glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-gamma-glutamate derivatives of folate cofactors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-gamma-glutamate and (4,4-difluoro)glutamyl-gamma-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a approximately 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is approximately 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under nonsaturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction.
Asunto(s)
Colorantes Fluorescentes/metabolismo , Péptidos/metabolismo , gamma-Glutamil Hidrolasa/metabolismo , Catálisis , Colorantes Fluorescentes/química , Hidrólisis , Cinética , Estructura Molecular , Especificidad por SustratoRESUMEN
Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Anilidas/farmacología , Inhibidores Enzimáticos/farmacología , Piperazinas/farmacología , Piperidinas/farmacología , Anilidas/química , Animales , Benzamidas/farmacología , Sitios de Unión/efectos de los fármacos , Carbamatos/farmacología , Inhibidores Enzimáticos/química , Humanos , Concentración 50 Inhibidora , Ratones , Piperazinas/química , Piperidinas/química , Serina/química , Factores de TiempoRESUMEN
How lipid transmitters move within and between cells to communicate signals remains an important and largely unanswered question. Integral membrane transporters, soluble lipid-binding proteins, and metabolic enzymes have all been proposed to collaboratively regulate lipid signaling dynamics in vivo. Assignment of the relative contributions made by each of these classes of proteins requires selective pharmacological agents to perturb their individual functions. Recently, LY2183240, a heterocyclic urea inhibitor of the putative endocannabinoid (EC) transporter, was shown to disrupt the cellular uptake of the lipid EC anandamide and promote analgesia in vivo. Here, we show that LY2183240 is a potent, covalent inhibitor of the EC-degrading enzyme fatty acid amide hydrolase (FAAH). LY2183240 inactivates FAAH by carbamylation of the enzyme's serine nucleophile. More global screens using activity-based proteomic probes identified several additional serine hydrolases that are also inhibited by LY2183240. These results indicate that the blockade of anandamide uptake observed with LY2183240 may be due primarily to the inactivation of FAAH, providing further evidence that this enzyme serves as a metabolic driving force that promotes the diffusion of anandamide into cells. More generally, the proteome-wide target promiscuity of LY2183240 designates the heterocyclic urea as a chemotype with potentially excessive protein reactivity for drug design.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Encéfalo/enzimología , Moduladores de Receptores de Cannabinoides/metabolismo , Endocannabinoides , Compuestos Heterocíclicos con 1 Anillo/química , Compuestos Heterocíclicos con 1 Anillo/farmacología , Hidrolasas/antagonistas & inhibidores , Urea/análogos & derivados , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Benzamidas/farmacología , Carbamatos/farmacología , Cisplatino , Eliminación de Gen , Ifosfamida , Masculino , Ratones , Ratones Endogámicos C57BL , Mitomicina , Estructura Molecular , Urea/química , Urea/farmacologíaRESUMEN
Fatty acid amide hydrolase (FAAH) regulates a large class of signaling lipids, including the endocannabinoid anandamide. Carbamate inhibitors of FAAH display analgesic and anxiolytic properties in rodents. However, the mechanism by which carbamates inhibit FAAH remains obscure. Here, we provide biochemical evidence that carbamates covalently modify the active site of FAAH by adopting an orientation opposite of that originally predicted from modeling. Based on these results, a series of carbamates was designed that display enhanced potency. One agent was converted into a "click chemistry" probe to comprehensively evaluate the proteome reactivity of FAAH-directed carbamates in vivo. These inhibitors were selective for FAAH in the nervous system, but they reacted with several enzymes in peripheral tissues. The experimental strategy described herein can be used to create in vivo probes for any enzyme susceptible to covalent inhibition.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Carbamatos/farmacología , Diseño de Fármacos , Sondas Moleculares/química , Amidohidrolasas/metabolismo , Animales , Carbamatos/química , Inhibidores Enzimáticos/química , Masculino , Ratones , Modelos Moleculares , Sondas Moleculares/farmacología , Estructura Molecular , ProteómicaRESUMEN
Acetaminophen (paracetamol) is a popular domestic analgesic and antipyretic agent with a weak anti-inflammatory action and a low incidence of adverse effects as compared with aspirin and other non-steroidal anti-inflammatory drugs. Here we show that acetaminophen, following deacetylation to its primary amine, is conjugated with arachidonic acid in the brain and the spinal cord to form the potent TRPV1 agonist N-arachidonoylphenolamine (AM404). This conjugation is absent in mice lacking the enzyme fatty acid amide hydrolase. AM404 also inhibits purified cyclooxygenase (COX)-1 and COX-2 and prostaglandin synthesis in lipopolysaccharide-stimulated RAW264.7 macrophages. This novel metabolite of acetaminophen also acts on the endogenous cannabinoid system, which, together with TRPV1 and COX, is present in the pain and thermoregulatory pathways. These findings identify fatty acid conjugation as a novel pathway for drug metabolism and provide a molecular mechanism for the occurrence of the analgesic N-acylphenolamine AM404 in the nervous system following treatment with acetaminophen.