Rat Fatty Acid Amide Hydrolase Research Articles

In Silico and In Vitro Analysis of Major Cannabis-Derived Compounds as Fatty Acid Amide Hydrolase Inhibitors.

Accumulated evidence suggests that enhancing the endocannabinoid (eCB) tone, in particular of anandamide (N-arachidonoylethanolamine, AEA), has therapeutic potential in many human diseases. Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme principally responsible for the degradation of AEA, and thus it represents a relevant target to increase signaling thereof. In recent years, different synthetic and natural compounds have been developed and tested on rat FAAH, but little is known of their effect on the human enzyme. Here, we sought to investigate six major cannabis-derived compounds to compare their action on rat and human FAAHs. To this aim, we combined an in silico analysis of their binding mode and affinity, with in vitro assays of their effect on enzyme activity. This integrated approach allowed to disclose differences in efficacy towards rat and human FAAHs, and to highlight the role of key residues involved in the inhibition of both enzymes. This study suggests that the therapeutic efficacy of compounds targeted towards FAAH should be always tested in vitro on both rat and human enzymes.

Design, microwave-assisted synthesis, biological evaluation and molecular modeling studies of 4-phenylthiazoles as potent fatty acid amide hydrolase inhibitors.

Endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are endogenous lipids that activate cannabinoid receptors. Activation of these receptors produces anti-inflammatory and analgesic effects. Fatty acid amide hydrolase (FAAH) is a membrane enzyme that hydrolases endocannabinoids; thus, inhibition of FAAH represents an attractive approach to develop new therapeutics for treating inflammation and pain. Previously, potent rat FAAH inhibitors containing 2-naphthyl- and 4-phenylthiazole scaffolds were identified, but up to the present time, very little structure-activity relationship studies have been performed on these moieties. We designed and synthesized several analogs containing these structural motifs and evaluated their inhibition potencies against human FAAH enzyme. In addition, we built and validated a homology model of human FAAH enzyme and performed docking experiments. We identified several inhibitors in the low nanomolar range and calculated their ADME predicted values. These FAAH inhibitors represent promising drug candidates for future preclinical in vivo studies.

Pharmacological characterization of a novel, potent, selective, and orally active fatty acid amide hydrolase inhibitor, PKM-833 [(R)-N-(pyridazin-3-yl)-4-(7-(trifluoromethyl)chroman-4-yl)piperazine-1-carboxamide] in rats: Potential for the treatment of inflammatory pain.

Recently, we identified a novel fatty acid amide hydrolase (FAAH) inhibitor, PKM‐833 [(R)‐N‐(pyridazin‐3‐yl)‐4‐(7‐(trifluoromethyl)chroman‐4‐yl)piperazine‐1‐carboxamide]. The aim of the present study is to characterize the pharmacological profile of PKM‐833 in vitro and in vivo. PKM‐833 showed potent inhibitory activities against human and rat FAAH with IC50 values of 8.8 and 10 nmol/L, respectively, 200‐fold more selectivity against other 137 molecular targets, and irreversible mode of action. In pharmacokinetic and pharmacodynamic studies, PKM‐833 showed excellent brain penetration and good oral bioavailability, and elevated anandamide (AEA) concentrations in the rat brain. These data indicate that PKM‐833 is a potent, selective, orally active, and brain‐penetrable FAAH inhibitor. In behavioral studies using rat models, PKM‐833 significantly attenuated formalin‐induced pain responses (3 mg/kg) and improved mechanical allodynia in complete freund's adjuvant (CFA)‐induced inflammatory pain (0.3‐3 mg/kg). On the other hand, PKM‐833 did not show the analgesic effects against mechanical allodynia in chronic constriction injury (CCI)‐induced neuropathic pain up to 30 mg/kg. Regarding side effects, PKM‐833 had no significant effects on catalepsy and motor coordination up to 30 mg/kg. These results indicate that PKM‐833 is a useful pharmacological agent that can be used to investigate the role of FAAH and may have therapeutic potential for the treatment of inflammatory pain without undesirable side effects.

An endocannabinoid catabolic enzyme FAAH and its paralogs in an early land plant reveal evolutionary and functional relationship with eukaryotic orthologs

Endocannabinoids were known to exist only among Animalia but recent report of their occurrence in early land plants prompted us to study its function and metabolism. In mammals, anandamide, as an endocannabinoid ligand, mediates several neurological and physiological processes, which are terminated by fatty acid amide hydrolase (FAAH). We identified nine orthologs of FAAH in the moss Physcomitrella patens (PpFAAH1 to PpFAAH9) with amidase signature and catalytic triad. The optimal amidase activity for PpFAAH1 was at 37 °C and pH 8.0, with higher specificity to anandamide. Further, the phylogeny and predicted structural analyses of the nine paralogs revealed that PpFAAH1 to PpFAAH4 were closely related to plant FAAH while PpFAAH6 to PpFAAH9 were to the rat FAAH, categorized based on the membrane binding cap, membrane access channel and substrate binding pocket. We also identified that a true ‘dynamic paddle’ that is responsible for tighter regulation of FAAH is recent in vertebrates and absent or not fully emerged in plants and non-vertebrates. These data reveal evolutionary and functional relationship among eukaryotic FAAH orthologs and features that contribute to versatility and tighter regulation of FAAH. Future studies will utilize FAAH mutants of moss to elucidate the role of anandamide in early land plants.

The endocannabinoid hydrolase FAAH is an allosteric enzyme

Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that in vivo controls content and biological activity of N-arachidonoylethanolamine (AEA) and other relevant bioactive lipids termed endocannabinoids. Parallel orientation of FAAH monomers likely allows both subunits to simultaneously recruit and cleave substrates. Here, we show full inhibition of human and rat FAAH by means of enzyme inhibitors used at a homodimer:inhibitor stoichiometric ratio of 1:1, implying that occupation of only one of the two active sites of FAAH is enough to fully block catalysis. Single W445Y substitution in rat FAAH displayed the same activity as the wild-type, but failed to show full inhibition at the homodimer:inhibitor 1:1 ratio. Instead, F432A mutant exhibited reduced specific activity but was fully inhibited at the homodimer:inhibitor 1:1 ratio. Kinetic analysis of AEA hydrolysis by rat FAAH and its F432A mutant demonstrated a Hill coefficient of ~1.6, that instead was ~1.0 in the W445Y mutant. Of note, also human FAAH catalysed an allosteric hydrolysis of AEA, showing a Hill coefficient of ~1.9. Taken together, this study demonstrates an unprecedented allosterism of FAAH, and represents a case of communication between two enzyme subunits seemingly controlled by a single amino acid (W445) at the dimer interface. In the light of extensive attempts and subsequent failures over the last decade to develop effective drugs for human therapy, these findings pave the way to the rationale design of new molecules that, by acting as positive or negative heterotropic effectors of FAAH, may control more efficiently its activity.

Comparison of Candida Albicans Fatty Acid Amide Hydrolase Structure with Homologous Amidase Signature Family Enzymes

Fatty acid amide hydrolase (FAAH) is a well-characterized member of the amidase signature (AS) family of serine hydrolases. The membrane-bound FAAH protein is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide and oleamide, that regulate a wide range of mammalian behaviors, including pain perception, inflammation, sleep, and cognitive/emotional state. To date, limited crystal structures of FAAH and non-mammalian AS family proteins have been determined and used for structure-based inhibitor design. In order to provide broader structural information, the crystal structure of FAAH from the pathogenic fungus Candida albicans was determined at a resolution of 2.2 Å. A structural comparison with a brown rat Rattus norvegicus FAAH as well as with other bacterial AS family members, MAE2 and PAM, showed overall similarities but there were several discriminative regions found: the transmembrane domain and the hydrophobic cap of the brown rat FAAH were completely absent in the fungal FAAH structure. Along with these results, a phylogenetic analysis of 19 species within the AS family showed that fungal FAAHs diverged from a common ancestor before the separation of eukarya and prokarya. Taken together, this study provides insights into developing more potent inhibitors of FAAH as well as expanding our knowledge of the relationships between AS family members.

Role of Steroids on the Membrane Binding Ability of Fatty Acid Amide Hydrolase.

Background: Fatty acid amide hydrolase (FAAH) is a membrane-bound homodimeric enzyme that gets in contact with a lipophilic substrate in the lipid bilayer, and then cleaves it into water soluble products. FAAH plays a critical role in modulating in vivo content and biological activity of endocannabinoids (eCBs), and its function is affected by membrane lipids. Increasing evidence suggests that also steroids can modulate endocannabinoid signaling, both in the central nervous system and at the periphery.Methods: In this study, we interrogated the effect of six steroids with relevant biological activity (testosterone, hydrocortisone, estradiol, pregnenolone, progesterone, and cortisone) on the membrane binding ability of rat FAAH. The experimental data analysis obtained by Fluorescence Resonance Energy Transfer Spectroscopy was paralleled by computational docking analysis.Results: Our data revealed distinct effects of the different steroids on the interaction of rat FAAH with model membranes. Among them, pregnenolone was found to be the most effective in raising rat FAAH affinity for model membranes. A possible binding pocket for steroid molecules was identified by docking analysis in the membrane-embedded region of the enzyme; such a pocket could account for the observed increase of the membrane affinity in the presence of the tested molecules.Conclusions: Overall, the results point to steroids as new regulators of FAAH interaction with membranes, which may impact the biological activity of eCBs.

Novel propanamides as fatty acid amide hydrolase inhibitors

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes.

Interaction of the N-(3-Methylpyridin-2-yl)amide Derivatives of Flurbiprofen and Ibuprofen with FAAH: Enantiomeric Selectivity and Binding Mode.

BackgroundCombined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.Methodology/Principal FindingsFAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAHT488A-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.Conclusions/SignificanceThe study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.

O-(triazolyl)methyl carbamates as a novel and potent class of fatty acid amide hydrolase (FAAH) inhibitors.

Inhibition of fatty acid amide hydrolase (FAAH) activity is under investigation as a valuable strategy for the treatment of several disorders, including pain and drug addiction. A number of potent FAAH inhibitors belonging to different chemical classes have been disclosed to date; O-aryl carbamates are one of the most representative families. In the search for novel FAAH inhibitors, a series of O-(1,2,3-triazol-4-yl)methyl carbamate derivatives were designed and synthesized exploiting a copper- catalyzed [3+2] cycloaddition reaction between azides and alkynes (click chemistry). Exploration of the structure-activity relationships within this new class of compounds identified potent inhibitors of both rat and human FAAH with IC50 values in the single-digit nanomolar range. In addition, these derivatives showed improved stability in rat plasma and kinetic solubility in buffer with respect to the lead compound. Based on the results of the study, the novel analogues identified can be considered to be promising starting point for the development of new FAAH inhibitors with improved drug-like properties.

Prostamide F(2) α receptor antagonism combined with inhibition of FAAH may block the pro-inflammatory mediators formed following selective FAAH inhibition.

Prostamides are lipid mediators formed by COX-2-catalysed oxidation of the endocannabinoid anandamide and eliciting effects often opposed to those caused by anandamide. Prostamides may be formed when hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) is physiologically, pathologically or pharmacologically decreased. Thus, therapeutic benefits of FAAH inhibitors might be attenuated by concomitant production of prostamide F2 α . This loss of benefit might be minimized by compounds designed to selectively antagonize prostamide receptors and also inhibiting FAAH. Inhibition of FAAH by a series of selective antagonists of prostamide receptors, including AGN 204396, AGN 211335 and AGN 211336, was assessed using rat, mouse and human FAAH in vitro, together with affinity for human recombinant CB1 and CB2 receptors. Effects in vivo were measured in a model of formalin-induced inflammatory pain in mice. The prostamide F2 α receptor antagonists were active against mouse and rat FAAH in the low μM range and behaved as non-competitive and plasma membrane-permeant inhibitors. AGN 211335, the most potent inhibitor of rat FAAH (IC50 = 1.2 μM), raised exogenous anandamide levels in intact cells and also bound to cannabinoid CB1 receptors. Both AGN 211335 and AGN 211336 (0.25-1 mg·kg(-1) , i.p.) inhibited the formalin-induced nociceptive response in mice. Synthetic compounds with indirect agonist activity at cannabinoid receptors and antagonist activity at prostamide receptors can be developed. Such compounds could be used as alternatives to selective FAAH inhibitors to prevent the possibility of prostamide F2 α -induced inflammation and pain. This article is part of a themed section on Cannabinoids 2013. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-6.

Rational design of fatty acid amide hydrolase inhibitors that act by covalently bonding to two active site residues.

The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.

Sulfonyl Fluoride Inhibitors of Fatty Acid Amide Hydrolase

Sulfonyl fluorides are known to inhibit esterases. Early work from our laboratory has identified hexadecyl sulfonylfluoride (AM374) as a potent in vitro and in vivo inhibitor of fatty acid amide hydrolase (FAAH). We now report on later generation sulfonyl fluoride analogs that exhibit potent and selective inhibition of FAAH. Using recombinant rat and human FAAH, we show that 5-(4-hydroxyphenyl)pentanesulfonyl fluoride (AM3506) has similar inhibitory activity for both the rat and the human enzyme, while rapid dilution assays and mass spectrometry analysis suggest that the compound is a covalent modifier for FAAH and inhibits its action in an irreversible manner. Our SAR results are highlighted by molecular docking of key analogs.

Rat and human fatty acid amide hydrolases: Overt similarities and hidden differences

Fatty acid amide hydrolase (FAAH) is a membrane protein that plays a relevant role in the metabolism of fatty acid amides and esters. It degrades important neurotransmitters such as oleamide and anandamide, and it has been involved in a number of human pathological conditions, representing therefore a valuable target for biochemical and pharmacological research. In this study, we have investigated in vitro the structure–function relationship of rat and human FAAHs. In particular circular dichroism, fluorescence spectroscopy and light scattering measurements have been performed, in order to characterize the structural features of the two proteins, both in the presence and absence of the irreversible inhibitor methoxyarachidonyl-fluorophosphonate. The results demonstrate that the structural dynamics of the two FAAHs are different, despite their high sequence homology and overall similarity in temperature-dependence. Additionally, membrane binding and kinetic assays of both FAAHs indicate that also the functional properties of the two enzymes are different in their interaction with lipid bilayers and with exogenous inhibitors. These findings suggest that pre-clinical studies of FAAH-dependent human diseases based only on animal models should be interpreted with caution, and that the efficacy of new drugs targeted to FAAH should be tested in vitro, on both rat and human enzymes.

2-Amino-5-arylbenzoxazole derivatives as potent inhibitors of fatty acid amide hydrolase (FAAH)

2,5-Disubstituted benzoxazole derivatives were evaluated for their ability to inhibit hFAAH. Structure–activity studies indicated that an isoindoline group at the 2-position of the benzoxazole ring gave rise to particularly potent inhibitors. Further refinement resulted in compounds, such as 50, with low nanomolar potencies against hFAAH. Preliminary biochemical experiments revealed that model benzoxazole FAAH inhibitors inhibited the enzyme in a manner consistent with a reversible mechanism. Additionally, the species dependency of FAAH inhibition was measured. Of the species tested, inhibition of rabbit FAAH, but not rat and guinea pig FAAH, appeared to show close alignment to human FAAH. These results may suggest similarities between the active sites of the FAAH enzyme for these two species, and may also suggest that rabbit could be a viable species with which to conduct preclinical testing with benzoxazole FAAH inhibitors.

Biochemical characterization and in vitro activity of AZ513, a noncovalent, reversible, and noncompetitive inhibitor of fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) hydrolyzes several bioactive lipids including the endocannabinoid anandamide. Synthetic FAAH inhibitors are being generated to help define the biological role(s) of this enzyme, the lipids it degrades in vivo, and the disease states that might benefit from its pharmacological modulation. AZ513 inhibits human FAAH (IC 50 = 551 nM), is 20-fold more potent against rat FAAH (IC 50 = 27 nM), and is inactive at 10 μM against the serine hydrolases acetylcholinesterase, thrombin, and trypsin. In contrast to most other potent FAAH inhibitors, AZ513 showed no evidence of covalently modifying the enzyme and displayed reversible inhibition. In an enzyme cross-competition assay, AZ513 did not compete with OL-135, an inhibitor that binds to the catalytic site in FAAH, which indicates that AZ513 does not bind to the catalytic site and is therefore noncompetitive with respect to substrate. AZ513 has good cell penetration as demonstrated by inhibition of anandamide hydrolysis in human FAAH-transfected HEK293 cells (IC 50 = 360 nM). AZ513 was tested in a rat spinal cord slice preparation where CB 1 activation reduces excitatory post-synaptic currents (EPSCs). In this native tissue assay of synaptic activity, AZ513 reduced EPSCs, which is consistent with inhibiting endogenous FAAH and augmenting endocannabinoid tone. AZ513 has a unique biochemical profile compared with other published FAAH inhibitors and will be a useful tool compound to further explore the role of FAAH in various biological processes.

Reversible Competitive α-Ketoheterocycle Inhibitors of Fatty Acid Amide Hydrolase Containing Additional Conformational Constraints in the Acyl Side Chain: Orally Active, Long-Acting Analgesics

A series of α-ketooxazoles containing conformational constraints in the C2 acyl side chain of 2 (OL-135) were examined as inhibitors of fatty acid amide hydrolase (FAAH). Only one of the two possible enantiomers displayed potent FAAH inhibition (S vs R enantiomer), and their potency is comparable or improved relative to 2, indicating that the conformational restriction in the C2 acyl side chain is achievable. A cocrystal X-ray structure of the α-ketoheterocycle 12 bound to a humanized variant of rat FAAH revealed its binding details, confirmed that the (S)-enantiomer is the bound active inhibitor, shed light on the origin of the enantiomeric selectivity, and confirmed that the catalytic Ser241 is covalently bound to the electrophilic carbonyl as a deprotonated hemiketal. Preliminary in vivo characterization of the inhibitors 12 and 14 is reported demonstrating that they raise brain anandamide levels following either intraperitoneal (ip) or oral (po) administration indicative of effective in vivo FAAH inhibition. Significantly, the oral administration of 12 caused dramatic accumulation of anandamide in the brain, with peak levels achieved between 1.5 and 3 h, and these elevations were maintained over 9 h. Additional studies of these two representative members of the series (12 and 14) in models of thermal hyperalgesia and neuropathic pain are reported, including the demonstration that 12 administered orally significantly attenuated mechanical (>6 h) and cold (>9 h) allodynia for sustained periods consistent with its long-acting effects in raising the endogenous concentration of anandamide.

Fluoride-mediated capture of a noncovalent bound state of a reversible covalent enzyme inhibitor: X-ray crystallographic analysis of an exceptionally potent α-ketoheterocycle inhibitor of fatty acid amide hydrolase.

Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (K(i) = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same "in action" state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.

Identification of potent, noncovalent fatty acid amide hydrolase (FAAH) inhibitors

Starting from a series of ureas that were determined to be mechanism-based inhibitors of FAAH, several spirocyclic ureas and lactams were designed and synthesized. These efforts identified a series of novel, noncovalent FAAH inhibitors with in vitro potency comparable to known covalent FAAH inhibitors. The mechanism of action for these compounds was determined through a combination of SAR and co-crystallography with rat FAAH.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.