RESUMEN

Fatty acid binding protein 5 (FABP5) is a promising target for development of inhibitors to help control pain and inflammation. In this work, computer-based docking (DOCK6 program) was employed to screen ∼2 M commercially available compounds to FABP5 based on an X-ray structure complexed with the small molecule inhibitor SBFI-26 previously identified by our group (also through virtual screening). The goal was discovery of additional chemotypes. The screen resulted in the purchase of 78 candidates, which led to the identification of a new inhibitor scaffold (STK-0) with micromolar affinity and apparent selectivity for FABP5 over FABP3. A second similarity-based screen resulted in three additional hits (STK-15, STK-21, STK-22) from which preliminary SAR could be derived. Notably, STK-15 showed comparable activity to the SBFI-26 reference under the same assay conditions (1.40 vs 0.86 µM). Additional molecular dynamics simulations, free energy calculations, and structural analysis (starting from DOCK-generated poses) revealed that R enantiomers (dihydropyrrole scaffold) of STK-15 and STK-22 have a more optimal composition of functional groups to facilitate additional H-bonds with Arg109 of FABP5. This observation suggests enantiomerically pure compounds could show enhanced activity. Overall, our study highlights the utility of using similarity-based screening methods to discover new inhibitor chemotypes, and the identified FABP5 hits provide a strong starting point for future efforts geared to improve activity.

Asunto(s)

Evaluación Preclínica de Medicamentos/métodos , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Supervivencia Celular/efectos de los fármacos , Cristalización , Cristalografía por Rayos X , Ciclobutanos/química , Ciclobutanos/farmacología , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/farmacología , Proteína 3 de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteína 3 de Unión a Ácidos Grasos/química , Células Endoteliales de la Vena Umbilical Humana , Humanos , Enlace de Hidrógeno , Ligandos , Simulación de Dinámica Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes/química , Interfaz Usuario-Computador

RESUMEN

(-)-Incarvillateine (INCA) is a natural product that has garnered attention due to its purported analgesic effects and historical use as a pain reliever in China. α-Truxillic acid monoesters (TAMEs) constitute a class of inhibitors targeting fatty acid binding protein 5 (FABP5), whose inhibition produces analgesia in animal models. The structural similarity between INCA and TAMEs motivated us to assess whether INCA exerts its antinociceptive effects via FABP inhibition. We found that, in contrast to TAMEs, INCA did not exhibit meaningful binding affinities toward four human FABP isoforms (FABP3, FABP4, FABP5 and FABP7) in vitro. INCA-TAME, a putative monoester metabolite of INCA that closely resembles TAMEs also lacked affinity for FABPs. Administration of INCA to mice produced potent antinociceptive effects while INCA-TAME was without effect. Surprisingly, INCA also potently suppressed locomotor activity at the same dose that produces antinociception. The motor suppressive effects of INCA were reversed by the adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine. Collectively, our results indicate that INCA and INCA-TAME do not inhibit FABPs and that INCA exerts potent antinociceptive and motor suppressive effects at equivalent doses. Therefore, the observed antinociceptive effects of INCA should be interpreted with caution.

Asunto(s)

Alcaloides/farmacología , Analgésicos/farmacología , Locomoción/efectos de los fármacos , Monoterpenos/farmacología , Nocicepción/efectos de los fármacos , Receptores de Adenosina A2/metabolismo , Agonistas del Receptor de Adenosina A2/farmacología , Antagonistas del Receptor de Adenosina A2/farmacología , Animales , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Masculino , Ratones , Unión Proteica , Teobromina/análogos & derivados , Teobromina/farmacología

RESUMEN

The increasing use of medical marijuana highlights the importance of developing a better understanding of cannabinoid metabolism. Phytocannabinoids, including ∆9-tetrahydrocannabinol (THC), are metabolized and inactivated by cytochrome P450 enzymes primarily within the liver. The lipophilic nature of cannabinoids necessitates mechanism(s) to facilitate their intracellular transport to metabolic enzymes. Here, we test the central hypothesis that liver-type fatty acid binding protein (FABP1) mediates phytocannabinoid transport and subsequent inactivation. Using X-ray crystallography, molecular modeling, and in vitro binding approaches we demonstrate that FABP1 accommodates one molecule of THC within its ligand binding pocket. Consistent with its role as a THC carrier, biotransformation of THC was reduced in primary hepatocytes obtained from FABP1-knockout (FABP1-KO) mice. Compared to their wild-type littermates, administration of THC to male and female FABP1-KO mice potentiated the physiological and behavioral effects of THC. The stark pharmacodynamic differences were confirmed upon pharmacokinetic analyses which revealed that FABP1-KO mice exhibit reduced rates of THC biotransformation. Collectively, these data position FABP1 as a hepatic THC transport protein and a critical mediator of cannabinoid inactivation. Since commonly used medications bind to FABP1 with comparable affinities to THC, our results further suggest that FABP1 could serve a previously unrecognized site of drug-drug interactions.

Asunto(s)

Dronabinol/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Animales , Sitios de Unión , Biotransformación , Células Cultivadas , Cristalografía por Rayos X , Dronabinol/administración & dosificación , Proteínas de Unión a Ácidos Grasos/química , Femenino , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares

RESUMEN

Fatty acid binding proteins (FABPs) serve as critical modulators of endocannabinoid signaling by facilitating the intracellular transport of anandamide and whose inhibition potentiates anandamide signaling. Our previous work has identified a novel small-molecule FABP inhibitor, α-truxillic acid 1-naphthyl monoester (SB-FI-26, 3) that has shown efficacy as an antinociceptive and anti-inflammatory agent in rodent models. In the present work, we have performed an extensive SAR study on a series of 3-analogs as novel FABP inhibitors based on computer-aided inhibitor drug design and docking analysis, chemical synthesis and biological evaluations. The prediction of binding affinity of these analogs to target FABP3, 5 and 7 isoforms was performed using the AutoDock 4.2 program, using the recently determined co-crystal structures of 3 with FABP5 and FABP7. The compounds with high docking scores were synthesized and evaluated for their activities using a fluorescence displacement assay against FABP3, 5 and 7. During lead optimization, compound 3l emerged as a promising compound with the Ki value of 0.21 µM for FABP 5, 4-fold more potent than 3 (Ki, 0.81 µM). Nine compounds exhibit similar or better binding affinity than 3, including compounds 4b (Ki, 0.55 µM) and 4e (Ki, 0.68 µM). Twelve compounds are selective for FABP5 and 7 with >10 µM Ki values for FABP3, indicating a safe profile to avoid potential cardiotoxicity concerns. Compounds 4f, 4j and 4k showed excellent selectivity for FABP5 and would serve as other new lead compounds. Compound 3a possessed high affinity and high selectivity for FABP7. Compounds with moderate to high affinity for FABP5 displayed antinociceptive effects in mice while compounds with low FABP5 affinity lacked in vivo efficacy. In vivo pain model studies in mice revealed that exceeding hydrophobicity significantly affects the efficacy. Thus, among the compounds with high affinity to FABP5 in vitro, the compounds with moderate hydrophobicity were identified as promising new lead compounds for the next round of optimization, including compounds 4b and 4j. For select cases, computational analysis of the observed SAR, especially the selectivity of new inhibitors to particular FABP isoforms, by comparing docking poses, interaction map, and docking energy scores has provided useful insights.

Asunto(s)

Analgésicos/farmacología , Ciclobutanos/farmacología , Ésteres/farmacología , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Analgésicos/síntesis química , Analgésicos/química , Animales , Diseño Asistido por Computadora , Ciclobutanos/síntesis química , Ciclobutanos/química , Relación Dosis-Respuesta a Droga , Diseño de Fármacos , Ésteres/síntesis química , Ésteres/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Simulación del Acoplamiento Molecular , Estructura Molecular , Relación Estructura-Actividad

RESUMEN

Endocannabinoids (eCBs) are lipid-signaling molecules involved in the regulation of numerous behaviors and physiological functions. Released by postsynaptic neurons, eCBs mediate retrograde modulation of synaptic transmission and plasticity by activating presynaptic cannabinoid receptors. While the cellular mechanisms by which eCBs control synaptic function have been well characterized, the mechanisms controlling their retrograde synaptic transport remain unknown. Here, we demonstrate that fatty-acid-binding protein 5 (FABP5), a canonical intracellular carrier of eCBs, is indispensable for retrograde eCB transport in the dorsal raphe nucleus (DRn). Thus, pharmacological inhibition or genetic deletion of FABP5 abolishes both phasic and tonic eCB-mediated control of excitatory synaptic transmission in the DRn. The blockade of retrograde eCB signaling induced by FABP5 inhibition is not mediated by impaired cannabinoid receptor function or reduced eCB synthesis. These findings indicate that FABP5 is essential for retrograde eCB signaling and may serve as a synaptic carrier of eCBs at central synapses.

Asunto(s)

Ácidos Araquidónicos/metabolismo , Endocannabinoides/farmacología , Proteínas de Unión a Ácidos Grasos/fisiología , Ácido Glutámico/metabolismo , Glicéridos/metabolismo , Proteínas de Neoplasias/fisiología , Sinapsis/fisiología , Transmisión Sináptica/efectos de los fármacos , Animales , Células Cultivadas , Endocannabinoides/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/metabolismo , Sinapsis/efectos de los fármacos

RESUMEN

Inhibition and genetic deletion of fatty acid-binding proteins (FABPs) 5 and 7 have been shown to increase the levels of the endocannabinoid anandamide as well as the related N-acylethanolamine's palmitoylethanolamide and oleoylethanolamide. This study examined the role of these FABPs on forced-swim (FS) behavior and on sucrose consumption in two experiments: (experiment 1) using wild-type (WT) mice treated with the FABP inhibitor SBFI26 or vehicle and (experiment 2) using WT and FABP5/7 deficient mice. Results from experiment 1 showed that acute treatment with SBFI26 did not have any effect on sucrose intake or FS behavior in mice. In experiment 2, male and female FABP5/7 deficient mice showed significant increases in sucrose consumption (25 and 21%, respectively) compared with their WT counterparts. In addition, immobility time during the FS was decreased by 27% in both male and female FABP5/7 knockout mice compared with their WT counterparts. The fact that such differences were seen between the acute pharmacological approach and the genetic approach (gene deletion) of FABP needs to be further investigated. The function of FABPs and their specific effects on endocannabinoid anandamide, oleoylethanolamide, and palmitoylethanolamide may play an important role in the development of reward and mood behaviors and could provide opportunities for potential therapeutic targets.

Asunto(s)

Proteína de Unión a los Ácidos Grasos 7/deficiencia , Proteínas de Unión a Ácidos Grasos/deficiencia , Preferencias Alimentarias/psicología , Reacción Cataléptica de Congelación/fisiología , Eliminación de Gen , Proteínas de Neoplasias/deficiencia , Sacarosa/metabolismo , Análisis de Varianza , Animales , Ácidos Araquidónicos/metabolismo , Ciclobutanos/farmacología , Ácidos Dicarboxílicos/farmacología , Endocannabinoides/metabolismo , Conducta Exploratoria/fisiología , Proteína de Unión a los Ácidos Grasos 7/genética , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Preferencias Alimentarias/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/genética , Ácidos Oléicos/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Factores Sexuales , Sacarosa/administración & dosificación , Natación/psicología

RESUMEN

Genetic and pharmacological manipulation of endocannabinoid (eCB) signaling has previously been shown to have an important role on the rewarding properties of drugs of abuse, including cocaine. Recently, fatty acid binding proteins (FABPs) have been proposed as intracellular transporters of the endocannabinoid anandamide (AEA) as well as other bioactive lipids to their catabolic enzyme, fatty acid amide hydrolase (FAAH). The role of these transporters in modulating the brains reward system has yet to be investigated. This study examined the effects of genetic deletion of FABP 5/7 on cocaine preference, as assessed by the Conditioned Place Preference (CPP) paradigm. Male and female wild type (WT) and FABP 5/7 KO mice showed similar acquisition of cocaine CPP, with no differences found in overall locomotor activity. In addition, while male and female WT mice showed stress-induced CPP for cocaine, male and female FABP 5/7 KO mice failed to show a stress-induced preference for the cocaine-paired chamber. Additionally, serum corticosterone levels were analyzed to explore any potential differences in stress response that may be responsible for the lack of stress-induced preference for cocaine. Serum samples were obtained in animals under basal conditions as well as following a 30-min tube restraint stress. Male and female FABP 5/7 KO mice showed reduced corticosterone levels under stress compared to their WT counterparts. The reduction in corticosterone response under stress may mediate that lack of a stress-induced preference for cocaine in the FABP 5/7 KO mice. Thus, the role of FABPs may play an important role in drug-seeking behavior under stressful conditions.

Asunto(s)

Cocaína , Corticosterona/sangre , Comportamiento de Búsqueda de Drogas/fisiología , Proteína de Unión a los Ácidos Grasos 7/deficiencia , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Neoplasias/deficiencia , Estrés Psicológico/sangre , Análisis de Varianza , Animales , Condicionamiento Operante/efectos de los fármacos , Comportamiento de Búsqueda de Drogas/efectos de los fármacos , Extinción Psicológica/fisiología , Proteína de Unión a los Ácidos Grasos 7/genética , Proteínas de Unión a Ácidos Grasos/genética , Femenino , Locomoción/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Neoplasias/genética , Recompensa , Estrés Psicológico/genética

RESUMEN

Human FABP5 and FABP7 are intracellular endocannabinoid transporters. SBFI-26 is an α-truxillic acid 1-naphthyl monoester that competitively inhibits the activities of FABP5 and FABP7 and produces antinociceptive and anti-inflammatory effects in mice. The synthesis of SBFI-26 yields several stereoisomers, and it is not known how the inhibitor binds the transporters. Here we report co-crystal structures of SBFI-26 in complex with human FABP5 and FABP7 at 2.2 and 1.9 Å resolution, respectively. We found that only (S)-SBFI-26 was present in the crystal structures. The inhibitor largely mimics the fatty acid binding pattern, but it also has several unique interactions. Notably, the FABP7 complex corroborates key aspects of the ligand binding pose at the canonical site previously predicted by virtual screening. In FABP5, SBFI-26 was unexpectedly found to bind at the substrate entry portal region in addition to binding at the canonical ligand-binding pocket. Our structural and binding energy analyses indicate that both R and S forms appear to bind the transporter equally well. We suggest that the S enantiomer observed in the crystal structures may be a result of the crystallization process selectively incorporating the (S)-SBFI-26-FABP complexes into the growing lattice, or that the S enantiomer may bind to the portal site more rapidly than to the canonical site, leading to an increased local concentration of the S enantiomer for binding to the canonical site. Our work reveals two binding poses of SBFI-26 in its target transporters. This knowledge will guide the development of more potent FABP inhibitors based upon the SBFI-26 scaffold.

Asunto(s)

Analgésicos/metabolismo , Ciclobutanos/metabolismo , Ácidos Dicarboxílicos/metabolismo , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Modelos Moleculares , Proteínas Supresoras de Tumor/metabolismo , Analgésicos/química , Analgésicos/farmacología , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/metabolismo , Antiinflamatorios no Esteroideos/farmacología , Apoproteínas/antagonistas & inhibidores , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Sitios de Unión , Dominio Catalítico , Biología Computacional , Cristalografía por Rayos X , Ciclobutanos/química , Ciclobutanos/farmacología , Ácidos Dicarboxílicos/química , Ácidos Dicarboxílicos/farmacología , Proteína de Unión a los Ácidos Grasos 7/antagonistas & inhibidores , Proteína de Unión a los Ácidos Grasos 7/química , Proteína de Unión a los Ácidos Grasos 7/genética , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Ligandos , Ratones , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas Recombinantes , Estereoisomerismo , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética

RESUMEN

Background Fatty-acid-binding proteins (FABPs) are intracellular carriers for endocannabinoids, N-acylethanolamines, and related lipids. Previous work indicates that systemically administered FABP5 inhibitors produce analgesia in models of inflammatory pain. It is currently not known whether FABP inhibitors exert their effects through peripheral or central mechanisms. Here, we examined FABP5 distribution in dorsal root ganglia and spinal cord and examined the analgesic effects of peripherally and centrally administered FABP5 inhibitors. Results Immunofluorescence revealed robust expression of FABP5 in lumbar dorsal root ganglia. FABP5 was distributed in peptidergic calcitonin gene-related peptide-expressing dorsal root ganglia and non-peptidergic isolectin B4-expressing dorsal root ganglia. In addition, the majority of dorsal root ganglia expressing FABP5 also expressed transient receptor potential vanilloid 1 (TRPV1) and peripherin, a marker of nociceptive fibers. Intraplantar administration of FABP5 inhibitors reduced thermal and mechanical hyperalgesia in the complete Freund's adjuvant model of chronic inflammatory pain. In contrast to its robust expression in dorsal root ganglia, FABP5 was sparsely distributed in the lumbar spinal cord and intrathecal administration of FABP inhibitor did not confer analgesic effects. Administration of FABP inhibitor via the intracerebroventricular (i.c.v.) route reduced thermal hyperalgesia. Antagonists of peroxisome proliferator-activated receptor alpha blocked the analgesic effects of peripherally and i.c.v. administered FABP inhibitor while antagonism of cannabinoid receptor 1 blocked the effects of peripheral FABP inhibition and a TRPV1 antagonist blocked the effects of i.c.v. administered inhibitor. Although FABP5 and TRPV1 were co-expressed in the periaqueductal gray region of the brain, which is known to modulate pain, knockdown of FABP5 in the periaqueductal gray using adeno-associated viruses and pharmacological FABP5 inhibition did not produce analgesic effects. Conclusions This study demonstrates that FABP5 is highly expressed in nociceptive dorsal root ganglia neurons and FABP inhibitors exert peripheral and supraspinal analgesic effects. This indicates that peripherally restricted FABP inhibitors may serve as a new class of analgesic and anti-inflammatory agents.

Asunto(s)

Analgésicos/uso terapéutico , Sistema Nervioso Central/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Proteínas de Neoplasias/metabolismo , Dolor/tratamiento farmacológico , Nervios Periféricos/metabolismo , Analgésicos/farmacología , Animales , Ácidos Araquidónicos/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Ciclobutanos/uso terapéutico , Ácidos Dicarboxílicos/uso terapéutico , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Adyuvante de Freund/toxicidad , Ganglios Espinales/metabolismo , Hiperalgesia/etiología , Inflamación/inducido químicamente , Inflamación/complicaciones , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas de Neoplasias/genética , Dolor/complicaciones , Dolor/etiología , Umbral del Dolor/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción Genética

RESUMEN

The endocannabinoid (eCB) system is involved in a wide range of behavioral disorders including alcoholism. Inhibition of fatty acid amide hydrolase (FAAH), the principal enzyme that degrades the eCB anandamide (AEA), which enhances AEA levels in the brain, significantly increases ethanol consumption and preference. In the present study, we examined whether pharmacological inhibition of fatty acid binding proteins (FABPs) 5 and 7, which blocks the transport of AEA to FAAH, and increase AEA levels in vivo also alters ethanol consumption and preference. Using a limited access two-bottle choice paradigm, we evaluated ethanol consumption in both male and female C57Bl/6 mice. Results showed a significant decrease in ethanol consumption in both males and females treated with SBFI26, an inhibitor of FABPs. Specifically, male and female mice treated with SBFI26 consumed 24% and 42% less compared to mice receiving no injections, respectively. Subsequently, corticosterone was examined to evaluate the effects FABP5/7 inhibition upon the stress response. We observed a significant elevation in corticosterone levels following restraint stress in SBFI26 treated females, with a weak effect seen in males as compared to vehicle. Based on our results, targeting of FABPs appears to play an important role in ethanol consumption that is differentially regulated in males and females, which is mediated by the stress response.

RESUMEN

This perspective was adapted from a Career Achievement Award talk given at the International Cannabinoid Research Society Symposium in Bukovina, Poland on June 27, 2016. As a biochemist working in the neurosciences, I was always fascinated with neurotransmitter inactivation. In 1993 we identified an enzyme activity that breaks down anandamide. We called the enzyme anandamide amidase, now called FAAH. We and other laboratories developed FAAH inhibitors that were useful reagents that also proved to have beneficial physiological effects and until recently, new generations of inhibitors were in clinical trials. Nearly all neurotransmitters are water soluble and as such, require a transmembrane protein transporter to pass through the lipid membrane for inactivation inside the cell. However, using model systems, we and others have shown that this is unnecessary for anandamide, an uncharged hydrophobic molecule that readily diffuses across the cellular membrane. Interestingly, its uptake is driven by the concentration gradient resulting from its breakdown mainly by FAAH localized in the endoplasmic reticulum. We identified the FABPs as intracellular carriers that "solubilize" anandamide, transporting anandamide to FAAH. Compounds that bind to FABPs block AEA breakdown, raising its level. The cannabinoids (THC and CBD) also were discovered to bind FABPs and this may be one of the mechanisms by which CBD works in childhood epilepsy, raising anandamide levels. Targeting FABPs may be advantageous since they have some tissue specificity and do not require reactive serine hydrolase inhibitors, as does FAAH, with potential for off-target reactions. At the International Cannabis Research Society Symposium in 1992, Raphe Mechoulam revealed that his laboratory isolated an endogenous lipid molecule that binds to the CB1 receptor (cannabinoid receptor type 1) and this became the milestone paper published in December of that year describing anandamide (AEA, Devane et al., 1992). As to be expected, this discovery raised the issues of AEA's synthesis and breakdown.

RESUMEN

The therapeutic properties of cannabinoids have been well demonstrated but are overshadowed by such adverse effects as cognitive and motor dysfunction, as well as their potential for addiction. Recent research on the natural lipid ligands of cannabinoid receptors, also known as endocannabinoids, has shed light on the mechanisms of intracellular transport of the endocannabinoid anandamide by fatty acid-binding proteins (FABPs) and subsequent catabolism by fatty acid amide hydrolase. These findings facilitated the recent development of SBFI26, a pharmacological inhibitor of epidermal- and brain-specific FABP5 and FABP7, which effectively increases anandamide signaling. The goal of this study was to examine this compound for any possible rewarding and addictive properties as well as effects on locomotor activity, working/recognition memory, and propensity for sociability and preference for social novelty (SN) given its recently reported anti-inflammatory and analgesic properties. Male C57BL mice were split into four treatment groups and conditioned with 5.0, 20.0, 40.0 mg/kg SBFI26, or vehicle during a conditioned place preference (CPP) paradigm. Following CPP, mice underwent a battery of behavioral tests [open field, novel object recognition (NOR), social interaction (SI), and SN] paired with acute SBFI26 administration. Results showed that SBFI26 did not produce CPP or conditioned place aversion regardless of dose and did not induce any differences in locomotor and exploratory activity during CPP- or SBFI26-paired open field activity. We also observed no differences between treatment groups in NOR, SI, and SN. In conclusion, as SBFI26 was shown previously by our group to have significant analgesic and anti-inflammatory properties, here we show that it does not pose a risk of dependence or motor and cognitive impairment under the conditions tested.

RESUMEN

Δ(9)-Tetrahydrocannabinol (THC) and cannabidiol (CBD) occur naturally in marijuana (Cannabis) and may be formulated, individually or in combination in pharmaceuticals such as Marinol or Sativex. Although it is known that these hydrophobic compounds can be transported in blood by albumin or lipoproteins, the intracellular carrier has not been identified. Recent reports suggest that CBD and THC elevate the levels of the endocannabinoid anandamide (AEA) when administered to humans, suggesting that phytocannabinoids target cellular proteins involved in endocannabinoid clearance. Fatty acid-binding proteins (FABPs) are intracellular proteins that mediate AEA transport to its catabolic enzyme fatty acid amide hydrolase (FAAH). By computational analysis and ligand displacement assays, we show that at least three human FABPs bind THC and CBD and demonstrate that THC and CBD inhibit the cellular uptake and catabolism of AEA by targeting FABPs. Furthermore, we show that in contrast to rodent FAAH, CBD does not inhibit the enzymatic actions of human FAAH, and thus FAAH inhibition cannot account for the observed increase in circulating AEA in humans following CBD consumption. Using computational molecular docking and site-directed mutagenesis we identify key residues within the active site of FAAH that confer the species-specific sensitivity to inhibition by CBD. Competition for FABPs may in part or wholly explain the increased circulating levels of endocannabinoids reported after consumption of cannabinoids. These data shed light on the mechanism of action of CBD in modulating the endocannabinoid tone in vivo and may explain, in part, its reported efficacy toward epilepsy and other neurological disorders.

Asunto(s)

Cannabidiol/metabolismo , Proteínas Portadoras/fisiología , Dronabinol/metabolismo , Proteínas de Unión a Ácidos Grasos/fisiología , Secuencia de Aminoácidos , Animales , Cannabidiol/química , Proteínas Portadoras/química , Dronabinol/química , Proteínas de Unión a Ácidos Grasos/química , Células HeLa , Humanos , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Ratas , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

The endocannabinoid anandamide (AEA) is an antinociceptive lipid that is inactivated through cellular uptake and subsequent catabolism by fatty acid amide hydrolase (FAAH). Fatty acid binding proteins (FABPs) are intracellular carriers that deliver AEA and related N-acylethanolamines (NAEs) to FAAH for hydrolysis. The mammalian brain expresses three FABP subtypes: FABP3, FABP5, and FABP7. Recent work from our group has revealed that pharmacological inhibition of FABPs reduces inflammatory pain in mice. The goal of the current work was to explore the effects of FABP inhibition upon nociception in diverse models of pain. We developed inhibitors with differential affinities for FABPs to elucidate the subtype(s) that contributes to the antinociceptive effects of FABP inhibitors. Inhibition of FABPs reduced nociception associated with inflammatory, visceral, and neuropathic pain. The antinociceptive effects of FABP inhibitors mirrored their affinities for FABP5, while binding to FABP3 and FABP7 was not a predictor of in vivo efficacy. The antinociceptive effects of FABP inhibitors were mediated by cannabinoid receptor 1 (CB1) and peroxisome proliferator-activated receptor alpha (PPARα) and FABP inhibition elevated brain levels of AEA, providing the first direct evidence that FABPs regulate brain endocannabinoid tone. These results highlight FABPs as novel targets for the development of analgesic and anti-inflammatory therapeutics.

Asunto(s)

Analgesia , Analgésicos/farmacología , Ácidos Araquidónicos/metabolismo , Encéfalo/metabolismo , Endocannabinoides/metabolismo , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Alcamidas Poliinsaturadas/metabolismo , Analgésicos/química , Analgésicos/metabolismo , Animales , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Ratones , Modelos Moleculares , Conformación Molecular , Simulación del Acoplamiento Molecular , Neuralgia/tratamiento farmacológico , Neuralgia/metabolismo , PPAR alfa/metabolismo , Unión Proteica , Ratas , Receptor Cannabinoide CB1/metabolismo

RESUMEN

In addition to binding intracellular fatty acids, fatty-acid-binding proteins (FABPs) have recently been reported to also transport the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG), arachidonic acid derivatives that function as neurotransmitters and mediate a diverse set of physiological and psychological processes. To understand how the endocannabinoids bind to FABPs, the crystal structures of FABP5 in complex with AEA, 2-AG and the inhibitor BMS-309403 were determined. These ligands are shown to interact primarily with the substrate-binding pocket via hydrophobic interactions as well as a common hydrogen bond to the Tyr131 residue. This work advances our understanding of FABP5-endocannabinoid interactions and may be useful for future efforts in the development of small-molecule inhibitors to raise endocannabinoid levels.

Asunto(s)

Ácidos Araquidónicos/química , Compuestos de Bifenilo/química , Endocannabinoides/química , Proteínas de Unión a Ácidos Grasos/química , Glicéridos/química , Proteínas de Neoplasias/química , Alcamidas Poliinsaturadas/química , Pirazoles/química , Animales , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Humanos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ratones , Modelos Moleculares , Proteínas de Neoplasias/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética

RESUMEN

The endocannabinoid system modulates numerous physiological processes including nociception and reproduction. Anandamide (AEA) is an endocannabinoid that is inactivated by cellular uptake followed by intracellular hydrolysis by fatty acid amide hydrolase (FAAH). Recently, FAAH-like anandamide transporter (FLAT), a truncated and catalytically-inactive variant of FAAH, was proposed to function as an intracellular AEA carrier and mediate its delivery to FAAH for hydrolysis. Pharmacological inhibition of FLAT potentiated AEA signaling and produced antinociceptive effects. Given that endocannabinoids produce analgesia through central and peripheral mechanisms, the goal of the current work was to examine the expression of FLAT in the central and peripheral nervous systems. In contrast to the original report characterizing FLAT, expression of FLAT was not observed in any of the tissues examined. To investigate the role of FLAT as a putative AEA binding protein, FLAT was generated from FAAH using polymerase chain reaction and further analyzed. Despite its low cellular expression, FLAT displayed residual catalytic activity that was sensitive to FAAH inhibitors and abolished following mutation of its catalytic serine. Overexpression of FLAT potentiated AEA cellular uptake and this appeared to be dependent upon its catalytic activity. Immunofluorescence revealed that FLAT localizes primarily to intracellular membranes and does not contact the plasma membrane, suggesting that its capability to potentiate AEA uptake may stem from its enzymatic rather than transport activity. Collectively, our data demonstrate that FLAT does not serve as a global intracellular AEA carrier, although a role in mediating localized AEA inactivation in mammalian tissues cannot be ruled out.

Asunto(s)

Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Endocannabinoides/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Amidohidrolasas/genética , Animales , Regulación Enzimológica de la Expresión Génica , Células HeLa , Humanos , Proteínas de Transporte de Membrana/genética , Ratones , Nervios Periféricos/enzimología , Transporte de Proteínas

RESUMEN

Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally referred to as Nacylethanolamines (NAEs). NAEs include anti-inflammatory and analgesic palmitoylethanolamide, anorexic oleoylethanolamide, and the endocannabinoid anandamide. Since the endogenous levels of NAEs are principally regulated by enzymes responsible for their biosynthesis and degradation, these enzymes are expected as targets for the development of therapeutic agents. Thus, a better understanding of these enzymes is indispensable. The classic "N-acylationphosphodiesterase pathway" for NAE biosynthesis is composed of two steps; the formation of Nacylphosphatidylethanolamine (NAPE) by N-acyltransferase and the release of NAE from NAPE by NAPE-hydrolyzing phospholipase D (NAPE-PLD). However, recent studies, including the analysis of NAPE-PLD-deficient (NAPE-PLD-/-) mice, revealed the presence of NAPE-PLD-independent multi-step pathways to form NAEs from NAPE in animal tissues. Our recent studies using NAPE-PLD-/- mice also suggest that NAE is formed not only from NAPE, but also from Nacylated plasmalogen-type ethanolamine phospholipid (N-acyl-plasmenylethanolamine) through both NAPE-PLDdependent and -independent pathways. Here, we present recent findings on NAE biosynthetic pathways mainly occurring in the brain.

Asunto(s)

Vías Biosintéticas/fisiología , Química Encefálica/fisiología , Encéfalo/metabolismo , Etanolaminas/síntesis química , Etanolaminas/metabolismo , Animales , Encéfalo/fisiología , Humanos

RESUMEN

Fatty acid binding proteins (FABPs), in particular FABP5 and FABP7, have recently been identified by us as intracellular transporters for the endocannabinoid anandamide (AEA). Furthermore, animal studies by others have shown that elevated levels of endocannabinoids resulted in beneficial pharmacological effects on stress, pain and inflammation and also ameliorate the effects of drug withdrawal. Based on these observations, we hypothesized that FABP5 and FABP7 would provide excellent pharmacological targets. Thus, we performed a virtual screening of over one million compounds using DOCK and employed a novel footprint similarity scoring function to identify lead compounds with binding profiles similar to oleic acid, a natural FABP substrate. Forty-eight compounds were purchased based on their footprint similarity scores (FPS) and assayed for biological activity against purified human FABP5 employing a fluorescent displacement-binding assay. Four compounds were found to exhibit approximately 50% inhibition or greater at 10 µM, as good as or better inhibitors of FABP5 than BMS309403, a commercially available inhibitor. The most potent inhibitor, γ-truxillic acid 1-naphthyl ester (ChemDiv 8009-2334), was determined to have K(i) value of 1.19±0.01 µM. Accordingly a novel α-truxillic acid 1-naphthyl mono-ester (SB-FI-26) was synthesized and assayed for its inhibitory activity against FABP5, wherein SB-FI-26 exhibited strong binding (K(i) 0.93±0.08 µM). Additionally, we found SB-FI-26 to act as a potent anti-nociceptive agent with mild anti-inflammatory activity in mice, which strongly supports our hypothesis that the inhibition of FABPs and subsequent elevation of anandamide is a promising new approach to drug discovery. Truxillic acids and their derivatives were also shown by others to have anti-inflammatory and anti-nociceptive effects in mice and to be the active component of Chinese a herbal medicine (Incarvillea sinensis) used to treat rheumatism and pain in humans. Our results provide a likely mechanism by which these compounds exert their effects.

Asunto(s)

Analgésicos/uso terapéutico , Antiinflamatorios/uso terapéutico , Ácidos Araquidónicos/metabolismo , Proteínas Portadoras/metabolismo , Endocannabinoides/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Analgésicos/química , Animales , Antiinflamatorios/química , Transporte Biológico , Descubrimiento de Drogas , Proteína de Unión a los Ácidos Grasos 7 , Humanos , Ratones

RESUMEN

We show that anandamide (AEA) externally added to model membrane vesicles containing trapped fatty acid amide hydrolyase (FAAH) can be readily hydrolyzed, demonstrating facile, rapid anandamide movement across the lipid bilayer. The rate of hydrolysis is significantly facilitated by cholesterol and coprostanol, but not by cholesterol sulfate. The effects of sterol upon hydrolysis by FAAH bound to the outer surface of the bilayer were much smaller, although they followed the same pattern. We propose the facilitation of hydrolysis is a combination of the effects of sterol on accessibility of membrane-inserted endocannabinoids to surface protein, and on the rate of endocannabinod transport across the membrane bilayer.

Asunto(s)

Amidohidrolasas/metabolismo , Ácidos Araquidónicos/metabolismo , Endocannabinoides/metabolismo , Liposomas/metabolismo , Alcamidas Poliinsaturadas/metabolismo , Esteroles/metabolismo , Amidohidrolasas/química , Ácidos Araquidónicos/química , Endocannabinoides/química , Hidrólisis , Liposomas/química , Membranas Artificiales , Alcamidas Poliinsaturadas/química , Esteroles/química

RESUMEN

N-acylethanolamines (NAEs) are bioactive lipids that engage diverse receptor systems. Recently, we identified fatty acid-binding proteins (FABPs) as intracellular NAE carriers. Here, we provide two new functions for FABPs in NAE signaling. We demonstrate that FABPs mediate the nuclear translocation of the NAE oleoylethanolamide, an agonist of nuclear peroxisome proliferator-activated receptor α (PPARα). Antagonism of FABP function through chemical inhibition, dominant-negative approaches, or shRNA-mediated knockdown reduced PPARα activation, confirming a requisite role for FABPs in this process. In addition, we show that NAE analogs, traditionally employed as inhibitors of the putative endocannabinoid transmembrane transporter, target FABPs. Support for the existence of the putative membrane transporter stems primarily from pharmacological inhibition of endocannabinoid uptake by such transport inhibitors, which are widely employed in endocannabinoid research despite lacking a known cellular target(s). Our approach adapted FABP-mediated PPARα signaling and employed in vitro binding, arachidonoyl-[1-(14)C]ethanolamide ([(14)C]AEA) uptake, and FABP knockdown to demonstrate that transport inhibitors exert their effects through inhibition of FABPs, thereby providing a molecular rationale for the underlying physiological effects of these compounds. Identification of FABPs as targets of transport inhibitors undermines the central pharmacological support for the existence of an endocannabinoid transmembrane transporter.

Asunto(s)

Núcleo Celular/metabolismo , Etanolaminas/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Oléicos/metabolismo , PPAR alfa/metabolismo , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular/fisiología , Animales , Moduladores de Receptores de Cannabinoides/genética , Moduladores de Receptores de Cannabinoides/metabolismo , Núcleo Celular/genética , Endocannabinoides , Proteínas de Unión a Ácidos Grasos/antagonistas & inhibidores , Proteínas de Unión a Ácidos Grasos/genética , Células HeLa , Humanos , Ratones , PPAR alfa/agonistas