Identification and optimization of soluble epoxide hydrolase inhibitors with dual potency towards fatty acid amide hydrolase

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.     

Synthesis of all-trans arachidonic acid and its effect on rabbit platelet aggregation

A simple and high-yielding method to convert natural all-cis PUFA derivatives to the corresponding all-trans geometrical isomers is described. The method is based on the thiyl radical-catalyzed cis-trans isomerization. The all-trans isomer of arachidonic acid was found to cause rabbit platelet aggregation at concentrations higher than 0.1 mM and inhibition of PAF-induced platelet aggregation in a concentration dependent manner with an IC(50) in the micromolar range.     

Novel inhibitors of fatty acid amide hydrolase

A class of bisarylimidazole derivatives are identified as potent inhibitors of the enzyme fatty acid amide hydrolase (FAAH). Compound 17 (IC(50)=2 nM) dose-dependently (0.1-10mg/kg, iv) potentiates the effects of exogenous anandamide (1 mg/kg, iv) in a rat thermal escape test (Hargreaves test), and shows robust antinociceptive activity in animal models of persistent (formalin test) and neuropathic (Chung model) pain. Compound 17 (20 mg/kg, iv) demonstrates activity in the formalin test that is comparable to morphine (3mg/kg, iv), and is dose-dependently inhibited by the CB1 antagonist SR141716A. In the Chung model, compound 17 shows antineuropathic effects similar to high-dose (100 mg/kg) gabapentin. FAAH inhibition shows potential utility for the clinical treatment of persistent and neuropathic pain.     

Hemodynamic profile, responsiveness to anandamide, and baroreflex sensitivity of mice lacking fatty acid amide hydrolase

The endocannabinoid anandamide exerts neurobehavioral, cardiovascular, and immune-regulatory effects through cannabinoid receptors (CB). Fatty acid amide hydrolase (FAAH) is an enzyme responsible for the in vivo degradation of anandamide. Recent experimental studies have suggested that targeting the endocannabinergic system by FAAH inhibitors is a promising novel approach for the treatment of anxiety, inflammation, and hypertension. In this study, we compared the cardiac performance of FAAH knockout (FAAH-/-) mice and their wild-type (FAAH+/+) littermates and analyzed the hemodynamic effects of anandamide using the Millar pressure-volume conductance catheter system. Baseline cardiovascular parameters, systolic and diastolic function at different preloads, and baroreflex sensitivity were similar in FAAH-/- and FAAH+/+ mice. FAAH-/- mice displayed increased sensitivity to anandamide-induced, CB1-mediated hypotension and decreased cardiac contractility compared with FAAH(+/+) littermates. In contrast, the hypotensive potency of synthetic CB1 agonist HU-210 and the level of expression of myocardial CB1 were similar in the two strains. The myocardial levels of anandamide and oleoylethanolamide, but not 2-arachidonylglycerol, were increased in FAAH-/- mice compared with FAAH+/+ mice. These results indicate that mice lacking FAAH have a normal hemodynamic profile, and their increased responsiveness to anandamide-induced hypotension and cardiodepression is due to the decreased degradation of anandamide rather than an increase in target organ sensitivity to CB1 agonists.     

The postmortal accumulation of brain N-arachidonylethanolamine (anandamide) is dependent upon fatty acid amide hydrolase activity

N-arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH-/-) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH-/- mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH-/- mice or mice treated with URB532. FAAH-/- mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.     

A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.     

Thiadiazolopiperazinyl ureas as inhibitors of fatty acid amide hydrolase

A series of thiadiazolopiperazinyl aryl urea fatty acid amide hydrolase (FAAH) inhibitors is described. The molecules were found to inhibit the enzyme by acting as mechanism-based substrates, forming a covalent bond with Ser241. SAR and PK properties are presented.     

N-acylethanolamine-hydrolyzing acid amidase and fatty acid amide hydrolase inhibition differentially affect N-acylethanolamine levels and macrophage activation

N-acylethanolamines (NAEs) such as N-palmitoylethanolamine and anandamide are endogenous bioactive lipids having numerous functions, including the control of inflammation. Their levels and therefore actions can be controlled by modulating the activity of two hydrolytic enzymes, N-acylethanolamine-hydrolyzing acid amidase (NAAA) and fatty acid amide hydrolase (FAAH). As macrophages are key to inflammatory processes, we used lipopolysaccharide-activated J774 macrophages, as well as primary mouse alveolar macrophages, to study the effect of FAAH and NAAA inhibition, using PF-3845 and AM9053 respectively, on macrophage activation and NAE levels measured by HPLC-MS. Markers of macrophage activation were measured by qRT-PCR and ELISA. Activation of macrophages decreased NAAA expression and NAE hydrolytic activity. FAAH and NAAA inhibition increased the levels of the different NAEs, although with different magnitudes, whether in control condition or following LPS-induced macrophage activation. Both inhibitors reduced several markers of macrophage activation, such as mRNA expression of inflammatory mediators, as well as cytokine and prostaglandin production, with however some differences between FAAH and NAAA inhibition. Most of the NAEs tested – including N-docosatetraenoylethanolamine and N-docosahexaenoylethanolamine – also reduced LPS-induced mRNA expression of inflammatory mediators, again with differences depending on the marker and the NAE, thus offering a potential explanation for the differential effect of the inhibitors on macrophage activation markers. In conclusion, we show different and complementary effects of NAE on lipopolysaccharide-induced macrophage activation. Our results support an important role for inhibition of NAE hydrolysis and NAAA inhibition in particular in controlling macrophage activation, and thus inflammation.     

Conformational effects in enzyme catalysis: reaction via a high energy conformation in fatty acid amide hydrolase

Quantum mechanics/molecular mechanics and molecular dynamics simulations of fatty acid amide hydrolase show that reaction (amide hydrolysis) occurs via a distinct, high energy conformation. This unusual finding has important implications for fatty acid amide hydrolase, a key enzyme in the endocannabinoid system. These results demonstrate the importance of structural fluctuations and the need to include them in the modeling of enzyme reactions. They also show that approaches based simply on studying enzyme-substrate complexes can be misleading for understanding biochemical reactivity.     

Development of highly sensitive fluorescent assays for fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) is a pharmaceutical target whose inhibition may lead to valuable therapeutics. Sensitive substrates for high-throughput assays are crucial for the rapid-screening FAAH inhibitors. Here we describe the development of novel and highly sensitive fluorescent assays for FAAH based on substituted aminopyridines. Examining the relationship between the structure and the fluorescence of substituted aminopyridines suggested that a methoxy group in the para position relative to the amino group in aminopyridines greatly increased the fluorescence (i.e., quantum yields approach unity). These novel fluorescent reporters had a high Stokes' shift of 94 nm, and their fluorescence in buffer systems increased with pH values from neutral to basic. Fluorescent substrates with these reporters displayed a very low fluorescent background and high aqueous solubility. Most importantly, fluorescent assays for FAAH based on these substrates were at least 25 times more sensitive than assays using related compounds with published colorimetric or fluorescent reporters. This property results in shorter assay times and decreased protein concentrations in the assays. Such sensitive assays will facilitate distinguishing the relative potency of powerful inhibitors of FAAH. When these fluorescent substrates were applied to human liver microsomes, results suggested that there was at least one amide hydrolase in addition to FAAH that could hydrolyze long-chain fatty acid amides. These results show that these fluorescent substrates are very valuable tools in FAAH activity assays including screening inhibitors by high-throughput assays instead of using the costly and labor-intensive radioactive ligands. Potential applications of novel fluorescent reporters are discussed.     

Heteroarylureas with spirocyclic diamine cores as inhibitors of fatty acid amide hydrolase

A series of mechanism based heteroaryl urea fatty acid amide hydrolase (FAAH) inhibitors with spirocyclic diamine cores is described. A potent member of this class, (37), was found to inhibit FAAH centrally, elevate the brain levels of three fatty acid ethanolamides [FAAs: anandamide (AEA), oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA)], and was moderately efficacious in a rat model of neuropathic pain.     

Novel mechanistic class of fatty acid amide hydrolase inhibitors with remarkable selectivity

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.     

Heterocyclic sulfoxide and sulfone inhibitors of fatty acid amide hydrolase

A novel series of heterocyclic sulfoxides and sulfones was prepared and examined as potential inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for inactivation of neuromodulating fatty acid amides including anandamide and oleamide.     

A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase

Fatty acid amide hydrolase (EC 3.5.1.4.) is the enzyme responsible for the rapid degradation of lipid-derived chemical messengers such as anandamide, oleamide, and 2-arachidonoylglycerol. The pharmacological characterization of this enzyme in vivo has been hampered by the lack of selective and bioavailable inhibitors. We have developed a simple, radioactive, high-throughput-compatible assay for this enzyme based on the differential absorption of the substrate and its products to activated charcoal. The assay was validated using known inhibitors. It may be applied for the identification of new inhibitors from a compound library.     

A second fatty acid amide hydrolase with variable distribution among placental mammals

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.     

A novel scintillation proximity assay for fatty acid amide hydrolase compatible with inhibitor screening

A binding assay for human fatty acid amide hydrolase (FAAH) using the scintillation proximity assay (SPA) technology is described. This SPA uses the specific interactions of [3H]R(+)-methanandamide (MAEA) and FAAH expressing microsomes to evaluate the displacement activity of FAAH inhibitors. We observed that a competitive nonhydrolyzed FAAH inhibitor, [3H]MAEA, bound specifically to the FAAH microsomes. Coincubation with an FAAH inhibitor, URB-597, competitively displaced the [3H]MAEA on the FAAH microsomes. The released radiolabel was then detected through an interaction with the SPA beads. The assay is specific for FAAH given that microsomes prepared from cells expressing the inactive FAAH-S241A mutant or vector alone had no significant ability to bind [3H]MAEA. Furthermore, the binding of [3H]MAEA to FAAH microsomes was abolished by selective FAAH inhibitors in a dose-dependent manner, with IC50 values comparable to those seen in a functional assay. This novel SPA has been validated and demonstrated to be simple, sensitive, and amenable to high-throughput screening.