Fatty acid amide hydrolase inhibitors display broad selectivity and inhibit multiple carboxylesterases as off-targets

Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug-drug interactions with other medicines if developed as therapeutic agents.     

Screening of curcumin‐derived isoxazole,pyrazoles, and pyrimidines for their anti‐inflammatory,antinociceptive, and cyclooxygenase‐2 inhibition

Curcumin has shown pharmacological properties against different phenotypes of various disease models. Different synthetic routes have been employed to develop its numerous derivatives for diverse and improved therapeutic roles. In this study, we have synthesized curcumin derivatives containing isoxazole, pyrazoles, and pyrimidines and then the synthesized molecules were evaluated for their anti‐inflammatory and antinociceptive activities in experimental animal models. Acute toxicity of synthesized molecules was evaluated in albino mice by oral administration. Any behavioral and neurological changes were observed at dose of 10 mg/kg body weight. Additionally, cyclooxygenase‐2 (COX‐2) enzyme inhibition studies were performed through in vitro assays. In vivo anti‐inflammatory studies showed that curcumin with pyrimidines was the most potent anti‐inflammatory agent which inhibited induced edema from 74.7% to 75.9%. Compounds 7 , 9 , and 12 exhibited relatively higher prevention of writhing episodes than any other compound with antinociceptive activity of 73.2%, 74.9%, and 71.8%, respectively. This was better than diclofenac sodium (reference drug, 67.1% inhibition). Similarly, COX‐2 in vitro inhibition assays results revealed that compound 12 (75.3% inhibition) was the most potent compound. Molecular docking studies of 10 , 11 , and 12 compounds in human COX‐2 binding site revealed the similar binding modes as that of other COX‐2‐selective inhibitors.     

Inhibition of ecto‐apyrase and ecto‐ATPase by pyridoxal phosphate‐related compounds

Studies of nucleotide receptors (P2‐receptors) in cells and tissues are complicated by cleavage of phosphate groups from nucleotide agonist ligands by ecto‐nucleotidases. Some P2 receptor antagonists may also inhibit ecto‐nucleotidases, making these studies even more complex. In order to systematically approach this problem, we investigated structure–activity relationships of pyridoxal‐5′‐phosphate‐6‐azophenyl‐2,4‐disulfonate (PPADS) and 14 derivatives, many potent as antagonists at P2 receptors, as inhibitors of ecto‐nucleotidases. The compounds were tested for their ability to inhibit enzymatic nucleotide breakdown by CHO cells stably transfected with plasmids containing the cDNA for rat ecto‐apyrase (NTPDase1) and rat ecto‐ATPase (NTPDase2). All inhibitors were tested at a concentration of 100 μM and ATP hydrolysis was quantified by HPLC. Maximal inhibition obtained for ecto‐apyrase and ecto‐ATPase was 60% and 35%, respectively. Most PPADS analogs were better inhibitors of ecto‐apyrase than of ecto‐ATPase. Compound 8, a phosphate derivative, inhibited ecto‐apyrase with no inhibition evident at ecto‐ATPase. Comparison of pharmacological data of PPADS analogs at P2 receptors as previously determined showed that four PPADS analogs exhibited selectivity for P2X nucleotide receptors. None of these compounds inhibited ecto‐ATPase, while two inhibited the ecto‐apyrase. Compound 14, a bisphosphate derivative, inhibited ecto‐ATPase without inhibition of ecto‐apyrase. This compound only weakly antagonized P2X₁ receptors and was inactive at P2X₂ and P2Y₁ receptors, thus bearing some selectivity for ecto‐ATPase. Compound 7, a 5‐methylphosphonate derivative, a potent antagonist of P2X₁ receptors, was inactive at ecto‐apyrase and only weakly inhibitory at ecto‐ATPase. Thus, PPADS modifications that enhance selectivity among ecto‐nucleotidases and P2 receptors have been identified. Drug Dev. Res. 51:153–158, 2000. Published 2001 Wiley‐Liss, Inc.     

FAAH and anandamide: is 2-AG really the odd one out?

Fatty acid amide hydrolase (FAAH) is a hydrolytic enzyme that recognizes as substrates and inactivates the two most studied endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG). Following the observation that endocannabinoids produced by tissues during pathological conditions often have protective roles, FAAH inhibitors have been proposed as therapeutic drugs. Yet it has been suggested that FAAH functions in vivo only as an anandamide-degrading enzyme because its pharmacological and genetic inactivation is usually accompanied by elevation of anandamide, but not 2-AG, levels. We believe, however, that this concept needs to be revisited in light of reports that, under certain experimental conditions, FAAH inhibitors also elevate 2-AG tissue levels in vivo and, more recently, that FAAH inactivation in the striatum instead reduces 2-AG concentrations through upregulation of anandamide levels, activation of transient receptor potential vanilloid 1 receptors and inhibition of 2-AG biosynthesis.     

Substituted 2-thioxoimidazolidin-4-ones and imidazolidine-2,4-diones as fatty acid amide hydrolase inhibitors templates

The demonstration of the essential role of fatty acid amide hydrolase (FAAH) in hydrolyzing endogenous bioactive fatty acid derivatives has launched the quest for the discovery of inhibitors for this enzyme. Along this line, a set of 58 imidazolidine-2,4-dione and 2-thioxoimidazolidin-4-one derivatives was evaluated as FAAH inhibitors. Among these compounds, 3-substituted 5,5'-diphenylimidazolidine-2,4-dione and 3-substituted 5,5'-diphenyl-2-thioxoimidazolidin-4-one derivatives were previously described as CB(1) cannabinoid receptor ligands. In the present study, we synthesized several derivatives exhibiting interesting FAAH inhibitory activity and devoid of affinity for the CB(1) and CB(2) cannabinoid receptors. For instance, 3-heptyl-5,5'-diphenylimidazolidine-2,4-dione (14) and 5,5'-diphenyl-3-tetradecyl-2-thioxo-imidazolidin-4-one (46) showed pI(50) values of 5.12 and 5.94, respectively. In conclusion, it appears that even though several 3-substituted 5,5'-diphenyl-2-thioxoimidazolidin-4-one and 3-substituted 5,5'-diphenylimidazolidine-2,4-dione derivatives have been previously shown to behave as CB(1) cannabinoid receptor ligands, appropriate substitutions of these templates can result in FAAH inhibitors devoid of affinity for the cannabinoid receptors.     

1,3‐Disubstituted‐4‐Aminopyrazolo [3, 4‐d] Pyrimidines,a New Class of Potent Inhibitors for Phospholipase D

Phospholipase D enzymes cleave lipid substrates to produce phosphatidic acid, an important precursor for many essential cellular molecules. Phospholipase D is a target to modulate cancer‐cell invasiveness. This study reports synthesis of a new class of phospholipase D inhibitors based on 1,3‐disubstituted‐4‐amino‐pyrazolopyrimidine core structure. These molecules were synthesized and used to perform initial screening for the inhibition of purified bacterial phospholipase D, which is highly homologous to the human PLD₁. Initially tested with the bacterial phospholipase D enzyme, then confirmed with the recombinant human PLD₁ and PLD₂ enzymes, the molecules presented here exhibited inhibition of phospholipase D activity (IC₅₀) in the low‐nanomolar to low‐micromolar range with both monomeric substrate diC₄PC and phospholipid vesicles and micelles. The data strongly indicate that these inhibitory molecules directly block enzyme/vesicle substrate binding. Preliminary activity studies using recombinant human phospholipase Ds in in vivo cell assays measuring both transphosphatidylation and head‐group cleavage indicate inhibition in the mid‐ to low‐nanomolar range for these potent inhibitory novel molecules in a physiological environment.     

Design, synthesis, and in vitro evaluation of carbamate derivatives of 2-benzoxazolyl- and 2-benzothiazolyl-(3-hydroxyphenyl)-methanones as novel fatty acid amide hydrolase inhibitors

Fatty acid amide hydrolase (FAAH) is an intracellular serine hydrolase, which catalyzes the hydrolysis of the endocannabinoid N-arachidonoylethanolamide to arachidonic acid and ethanolamine. FAAH also hydrolyzes another endocannabinoid, 2-arachidonoylglycerol (2-AG). However, 2-AG has been assumed to be hydrolyzed mainly by monoacylglycerol lipase (MAGL) or a MAGL-like enzyme. Inhibition of FAAH or MAGL activity might lead to beneficial effects in many physiological disorders such as pain, inflammation, and anxiety due to increased endocannabinoid-induced activation of cannabinoid receptors CB1 and CB2. In the present study, a total of 34 novel compounds were designed, synthesized, characterized, and tested against FAAH and MAGL-like enzyme activity. Altogether, 16 compounds were found to inhibit FAAH with half-maximal inhibition concentrations (IC50) between 28 and 380 nM. All the active compounds belong to the structural family of carbamates. Compounds 14 and 18 were found to be the most potent FAAH inhibitors, which may serve as lead structures for novel FAAH inhibitors.     

Reduced anxiety-like behaviour induced by genetic and pharmacological inhibition of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) is mediated by CB1 receptors

Anandamide and 2-arachidonoyl glycerol, referred to as endocannabinoids (eCBs), are the endogenous agonists for the cannabinoid receptor type 1 (CB1). Several pieces of evidence support a role for eCBs in the attenuation of anxiety-related behaviours, although the precise mechanism has remained uncertain. The fatty acid amid hydrolase (FAAH), an enzyme responsible for the degradation of eCBs, has emerged as a promising target for anxiety-related disorders, since FAAH inhibitors are able to increase the levels of anandamide and thereby induce anxiolytic-like effects in rodents. The present study adopted both genetic and pharmacological approaches and tested the hypothesis that FAAH-deficient (FAAH(-/-)) mice as well as C57BL/6N mice treated with an FAAH inhibitor (URB597) would express reduced anxiety-like responses. Furthermore, as it is known that anandamide can bind several other targets than CB1 receptors, we investigated whether FAAH inhibition reduces anxiety via CB1 receptors. FAAH(-/-) mice showed reduced anxiety both in the elevated plus maze and in the light-dark test. These genotype-related differences were prevented by the CB1 receptor antagonist rimonabant (3mg/kg). Moreover, URB597 (1mg/kg) induced an anxiolytic-like effect in C57BL/6N mice exposed to the elevated plus maze, which was prevented by rimonabant (3mg/kg). The present work provides genetic and pharmacological evidence supporting the inhibition of FAAH as an important mechanism for the alleviation of anxiety. In addition, it indicates an increased activation of CB1 receptors as a mechanism underlying the effects of FAAH inhibition in two models of anxiety.     

Molecular basis for covalent inhibition of glyceraldehyde‐3‐phosphate dehydrogenase by a 2‐phenoxy‐1,4‐naphthoquinone small molecule

Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) has recently gained attention as an antiprotozoan and anticancer drug target. We have previously identified 2‐phenoxy‐1,4‐naphthoquinone as an inhibitor of both Trypanosoma brucei and human GAPDH. Herein, through multiple chemical, biochemical, and biological studies, and through the design of analogs, we confirmed the formation of a covalent adduct, we clarified the inhibition mechanism, and we demonstrated antitrypanosomal, antiplasmodial, and cytotoxic activities in cell cultures. The overall results lent support to the hypothesis that 2‐phenoxy‐1,4‐naphthoquinone binds the GAPDH catalytic cysteine covalently through a phenolate displacement mechanism. By investigating the reactivity of 2‐phenoxy‐1,4‐naphthoquinone and its analogs with four GAPDH homologs, we showed that the covalent inhibition is not preceded by the formation of a strong non‐covalent complex. However, an up to fivefold difference in inactivation rates among homologs hinted at structural or electrostatic differences of their active sites that could be exploited to further design kinetically selective inhibitors. Moreover, we preliminarily showed that 2‐phenoxy‐1,4‐naphthoquinone displays selectivity for GAPDHs over two other cysteine‐dependent enzymes, supporting its suitability as a warhead starting fragment for the design of novel inhibitors.     

The effects of jatrorrhizine on contractile responses of rat ileum

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.     

Fatty acid amide hydrolase inhibitors from virtual screening of the endocannabinoid system

The endocannabinoid system consists of two cannabinoid receptors (CB1 and CB2), endogenous ligands (endocannabinoids), and the enzymes involved in the metabolism of the endocannabinoids, including fatty acid amide hydrolase (FAAH) and monoglyceride lipase (MGL). In the present study, virtual screening of MGL inhibitors was performed by utilizing a comparative model of the human MGL enzyme. All hit molecules were tested for their potential MGL inhibitory activity, but no compounds were found capable of inhibiting MGL-like enzymatic activity in rat cerebellar membranes. However, these compounds were also tested for their potential FAAH inhibitory activity and five compounds (2-6) inhibiting FAAH were found with IC50 values between 4 and 44 microM. In addition, the hit molecules from the virtual screening of CB2 receptor ligands (reported previously in Salo et al. J. Med. Chem. 2005, 48, 7166) were also tested in our FAAH assay, and four active compounds (7-10) were found with IC50 values between 0.52 and 22 microM. Additionally, compound 7 inhibited MGL-like enzymatic activity with an IC50 value of 31 microM.     

Pyridine‐substituted thiazolylphenol derivatives: Synthesis,modeling studies,aromatase inhibition,and antiproliferative activity evaluation

Drugs used in breast cancer treatments target the suppression of estrogen biosynthesis. During this suppression, the main goal is to inhibit the aromatase enzyme that is responsible for the cyclization and structuring of estrogens either with steroid or non‐steroidal‐type inhibitors. Non‐steroidal derivatives generally have a planar aromatic structure attached to the triazole ring system in their structures, which inhibits hydroxylation reactions during aromatization by coordinating the heme group. Bioisosteric replacement of the triazole ring system and development of aromatic/cyclic structures of the side chain can increase the selectivity for aromatase enzyme inhibition. In this study, pyridine‐substituted thiazolylphenol derivatives, which are non‐steroidal triazole bioisosteres, were synthesized using the Hantzsch method, and physical analysis and structural determination studies were performed. The IC₅₀ values of the compounds were determined by a fluorescence‐based aromatase inhibition assay. Then, their antiproliferative activities on the MCF7 and HEK 293 cell lines were evaluated with the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. Furthermore, the crystal structure of human placental aromatase was subjected to a series of docking experiments to identify the possible interactions between the most active structure and the active site. Lastly, an in silico technique was performed to analyze and predict the drug‐likeness, molecular and ADME properties of the synthesized molecules.

     

Monoglyceride lipase-like enzymatic activity is responsible for hydrolysis of 2-arachidonoylglycerol in rat cerebellar membranes

2-Arachidonoylglycerol (2-AG) is an endogenous cannabinoid that binds to CB1 and CB2 cannabinoid receptors, inducing cannabimimetic effects. However, the cannabimimetic effects of 2-AG are weak in vivo due to its rapid enzymatic hydrolysis. The enzymatic hydrolysis of 2-AG has been proposed to mainly occur by monoglyceride lipase (monoacylglycerol lipase). Fatty acid amide hydrolase (FAAH), the enzyme responsible for the hydrolysis of N-arachidonoylethanolamide (AEA), is also able to hydrolyse 2-AG. In the present study, we investigated the hydrolysis of endocannabinoids in rat cerebellar membranes and observed that enzymatic activity towards 2-AG was 50-fold higher than that towards AEA. Furthermore, various inhibitors for 2-AG hydrolase activity were studied in rat cerebellar membranes. 2-AG hydrolysis was inhibited by methyl arachidonylfluorophosphonate, hexadecylsulphonyl fluoride and phenylmethylsulphonyl fluoride with ic(50) values of 2.2 nM, 241 nM and 155 microM, respectively. Potent FAAH inhibitors, such as OL-53 and URB597, did not inhibit the hydrolysis of 2-AG, suggesting that 2-AG is inactivated in rat cerebellar membranes by an enzyme distinct of FAAH. The observation that the hydrolysis of 1(3)-AG and 2-AG occurred at equal rates supports the role of MGL in 2-AG inactivation. This enzyme assay provides a useful method for future inhibition studies of 2-AG degrading enzyme(s) in brain membrane preparation having considerably higher MGL-like activity when compared to FAAH activity.     

SAR and LC/MS studies of β-lactamic inhibitors of human fatty acid amide hydrolase (hFAAH): evidence of a nonhydrolytic process

The endocannabinoid hydrolyzing enzyme FAAH uses a nonclassical catalytic triad (namely, Ser-Ser-Lys instead of Ser-Asp-His) to cleave its endogenous substrates. Because inhibiting FAAH has a clear therapeutic potential, we previously developed β-lactam-type inhibitors of hFAAH. Here, we report the synthesis of five novel derivatives (5-9) of our lead compound 1-(pent-4-enoyl)-3(S)-[1(R)-(4-phenylbutanoyloxy)-ethyl]-azetidin-2-one (4, IC(50) = 5 nM) obtained via the systematic replacement of one to three carbonyls by methylene groups. The SAR results showed that the imide, but not the lactam, function is essential to the inhibition of hFAAH. We also performed LC/MS analysis following incubation of our inhibitors with hFAAH or mouse liver. We demonstrated that hFAAH interacts with these β-lactam-type inhibitors but, unexpectedly, does not open the β-lactam moiety. This mechanism seems to be unique to FAAH because the β-lactam function of the inhibitors is hydrolyzed when they are incubated in the presence of the serine hydrolases expressed in the mouse liver. Finally, we confirmed these results by showing that a highly selective FAAH inhibitor (PF-750) does not prevent this hydrolysis by liver homogenates.     

Evaluation of Imidazole‐Based Compounds as Heme Oxygenase‐1 Inhibitors

Imidazole‐based compounds previously synthesized in our laboratory were selected and reconsidered as inhibitors of heme oxygenase‐1 obtained from the microsomal fractions of rat spleens. Most of tested compounds were good inhibitors with IC₅₀ values in the low micromolar range. Compounds were also assayed on membrane‐free full‐length recombinant human heme oxygenase‐1; all tested compounds were unable to interact with human heme oxygenase‐1 at 100 μm concentrations with the exception of compounds 11 and 13 that inhibited the enzyme of 54% and 20%, respectively. The binding of the most active compound 11 with heme or heme‐conjugated human heme oxygenase‐1 was also examined by spectral analyses. When heme was not conjugated to human heme oxygenase‐1, compound 11 caused changes in the heme spectrum only at concentration 50‐fold (100 μm ) higher than that required to inhibit rat heme oxygenase‐1; when heme was conjugated to human heme oxygenase‐1, compound 11 was able to form a heme‐compound 11 complex also at low micromolar concentrations. To obtain information on the binding mode of the tested compounds with enzyme, docking studies and pharmacophore analysis were performed. Template docking results were in agreement with experimental inhibition data and with a structure‐based pharmacophoric model. These data may be exploitable to design new OH‐1 inhibitors.     

Discovery of potent inhibitors of human and mouse Fatty Acid amide hydrolases

Fatty acid amide hydrolase (FAAH, EC 3.5.1.99) is the main enzyme catabolizing endocannabinoid fatty acid amides. FAAH inactivation promotes beneficial effects upon pain and anxiety without the side effects accompanying agonists of type-1 cannabinoid receptors. Aiming at discovering new selective FAAH inhibitors, we developed a series of compounds (5a-u) characterized by a functionalized heteroaromatic scaffold. Particularly, 5c and 5d were identified as extremely potent, noncompetitive, and reversible FAAH inhibitors endowed with a remarkable selectivity profile and lacking interaction with the hERG channels. In vivo antinociceptive activity was demonstrated for 5c, 5d, and 5n at a dose much lower than that able to induce either striatal and limbic stereotypies or anxiolytic activity, thus outlining their potential to turn into optimum preclinical candidates. Aiming at improving pharmacokinetic properties and metabolic stability of 5d, we developed a subset of nanomolar dialyzable FAAH inhibitors (5v-z), functionalized by specific polyethereal lateral chains and fluorinated aromatic rings.     

Exploration of 5‐(5‐nitrothiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazoles as selective,multitargeted antimycobacterial agents

We report the biological evaluation of 5‐(5‐nitrothiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazole derivatives against bacteria, eukaryotic cell lines and the assessment of their mechanisms of action to determine their prospects of being developed into potent antituberculosis agents. The compounds were evaluated for their antibacterial property against Mycobacterium tuberculosis H37Rv, multidrug‐resistant M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus using high‐throughput spot‐culture growth inhibition assay. They were found to be selective toward slow‐growing mycobacteria and Gram‐positive bacteria. In M. bovis BCG, they exhibited a bactericidal mode of action. Cytotoxicity was assessed in human THP‐1 and murine RAW 264.7 cell lines, and the compounds showed a lower cytotoxicity potential when compared with their antibacterial activity. They were found to be excellent whole‐cell efflux pump inhibitors of the mycobacterial surrogate M. aurum, performing better than known efflux pump inhibitor verapamil. The 5‐nitrothiophene moiety was identified for the first time as a prospective inhibitor scaffold of mycobacterial arylamine N‐acetyltransferase enzyme, which is the key enzyme in metabolizing isoniazid, a first‐line antituberculosis drug. The two aforementioned findings make the compounds potential hits in the development of adjunctive tuberculosis therapy.