Fatty-acid-binding protein 5 controls retrograde endocannabinoid signaling at central glutamate synapses.

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.

Identification of Fatty Acid Binding Protein 5 Inhibitors Through Similarity-Based Screening.

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.

Fatty acid binding protein deletion prevents stress-induced preference for cocaine and dampens stress-induced corticosterone levels.

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.

Fatty acid-binding proteins 5 and 7 gene deletion increases sucrose consumption and diminishes forced swim immobility time.

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.

The Antinociceptive Agent SBFI-26 Binds to Anandamide Transporters FABP5 and FABP7 at Two Different Sites.

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.

Fatty-acid-binding protein inhibition produces analgesic effects through peripheral and central mechanisms.

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.

Fatty acid-binding proteins (FABPs) are intracellular carriers for Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD).

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.

Crystallographic study of FABP5 as an intracellular endocannabinoid transporter.

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.

Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors.

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.

Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways.

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg(2+)-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.

Studies on the anorectic effect of N-acylphosphatidylethanolamine and phosphatidylethanolamine in mice.

N-acyl-phosphatidylethanolamine is a precursor phospholipid for anandamide, oleoylethanolamide, and other N-acylethanolamines, and it may in itself have biological functions in cell membranes. Recently, N-palmitoyl-phosphatidylethanolamine (NAPE) has been reported to function as an anorectic hormone secreted from the gut and acting on the brain (Gillum et al., [5]). In the current study, two of our laboratories independently investigated whether NAPE metabolites may be involved in mediating the anorectic action of NAPE i.p. injected in mice. Thus, the anorectic activity of a non-hydrolysable NAPE analogue, having ether bonds instead of ester bonds at sn1 and sn2 was compared with that of NAPE in molar equivalent doses. Furthermore, the anorectic effect of NAPE in NAPE-hydrolysing phospholipase D knockout animals was investigated. As negative controls, the NAPE precursor phosphatidylethanolamine and the related phospholipids phosphatidylcholine and phosphatidic acid were also tested. All compounds except one were found to inhibit food intake, raising the possibility that the effect of NAPE is non-specific.

Identification of intracellular carriers for the endocannabinoid anandamide.

The endocannabinoid anandamide (arachidonoyl ethanolamide, AEA) is an uncharged neuromodulatory lipid that, similar to many neurotransmitters, is inactivated through its cellular uptake and subsequent catabolism. AEA is hydrolyzed by fatty acid amide hydrolase (FAAH), an enzyme localized on the endoplasmic reticulum. In contrast to most neuromodulators, the hydrophilic cytosol poses a diffusional barrier for the efficient delivery of AEA to its site of catabolism. Therefore, AEA likely traverses the cytosol with the assistance of an intracellular carrier that increases its solubility and rate of diffusion. To study this process, AEA uptake and hydrolysis were examined in COS-7 cells expressing FAAH restricted to the endoplasmic reticulum, mitochondria, or the Golgi apparatus. AEA hydrolysis was detectable at the earliest measurable time point (3 seconds), suggesting that COS-7 cells, normally devoid of an endocannabinoid system, possess an efficient cytosolic trafficking mechanism for AEA. Three fatty acid binding proteins (FABPs) known to be expressed in brain were examined as possible intracellular AEA carriers. AEA uptake and hydrolysis were significantly potentiated in N18TG2 neuroblastoma cells after overexpression of FABP5 or FABP7, but not FABP3. Similar results were observed in COS-7 cells stably expressing FAAH. Consistent with the roles of FABP as AEA carriers, administration of the competitive FABP ligand oleic acid or the selective non-lipid FABP inhibitor BMS309403 attenuated AEA uptake and hydrolysis by approximately 50% in N18TG2 and COS-7 cells. Taken together, FABPs represent the first proteins known to transport AEA from the plasma membrane to FAAH for inactivation and may therefore be novel pharmacological targets.

Lipid droplets are novel sites of N-acylethanolamine inactivation by fatty acid amide hydrolase-2.

Anandamide (AEA) and other bioactive N-acylethanolamines (NAEs) are primarily inactivated by the enzyme fatty acid amide hydrolase (FAAH). Recently, FAAH-2 was discovered in humans, suggesting an additional enzyme can mediate NAE inactivation in higher mammals. Here, we performed a biochemical characterization of FAAH-2 and explored its capacity to hydrolyze NAEs in cells. In homogenate activity assays, FAAH-2 hydrolyzed AEA and palmitoylethanolamide (PEA) with activities approximately 6 and approximately 20% those of FAAH, respectively. In contrast, FAAH-2 hydrolyzed AEA and PEA in intact cells with rates approximately 30-40% those of FAAH, highlighting a potentially greater contribution toward NAE catabolism in vivo than previously appreciated. In contrast to endoplasmic reticulum-localized FAAH, immunofluorescence revealed FAAH-2 was localized on lipid droplets. Supporting this distribution pattern, the putative N-terminal hydrophobic region of FAAH-2 was identified as a functional lipid droplet localization sequence. Lipid droplet localization was essential for FAAH-2 activity as chimeras excluded from lipid droplets lacked activity and/or were poorly expressed. Lipid droplets represent novel sites of NAE inactivation. Therefore, we examined substrate delivery to these organelles. AEA was readily trafficked to lipid droplets, confirming that lipid droplets constitute functional sites of NAE inactivation. Collectively, these results establish FAAH-2 as a bone fide NAE-catabolizing enzyme and suggest that NAE inactivation is spatially separated in cells of higher mammals.

Incarvillateine produces antinociceptive and motor suppressive effects via adenosine receptor activation.

(-)-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.

FABP1 controls hepatic transport and biotransformation of Δ9-THC.

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.

SAR studies on truxillic acid mono esters as a new class of antinociceptive agents targeting fatty acid binding proteins.

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.

Pharmacological Inhibition of Brain Fatty Acid Binding Protein Reduces Ethanol Consumption in Mice.

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.

Design of on-target FAAH inhibitors.

In this issue of Chemistry & Biology, Alexander and Cravatt propose a model for the binding of carbamate inhibitors to fatty acid amide hydrolase (FAAH), the enzyme that breaks down signaling lipids. Using competitive activity-based protein profiling and click chemistry, they designed potent and selective FAAH inhibitors and characterized their off-target reactions.

A Personal Retrospective: Elevating Anandamide (AEA) by Targeting Fatty Acid Amide Hydrolase (FAAH) and the Fatty Acid Binding Proteins (FABPs).

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.