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.     

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.     

Benzoquinones as inhibitors of botulinum neurotoxin serotype A

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC₅₀ values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC₅₀ competitive inhibitors that depend on the BQ moiety for activity.     

SAR studies of 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one as inhibitors of 4-hydroxyphenylpyruvate dioxygenase

Various 3-cyclopropanecarbonyloxy-2-cyclohexen-1-one 1 derivatives have been synthesized and tested as inhibitors of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) from pig liver. The inhibition results indicated that well-positioned dicarbonyl groups as well as the cyclopropyl group of 1 were essential for potent inhibition. Substitution at the 2-position of the ring system has a significant effect on inhibitor potency, while the 5-position can undergo substantial variations and retain inhibitor potency. In the compounds examined, 2-chloro substituted 12 is the best inhibitor of all with IC(50) of 15 nM, the rest of the synthesized analogues were less potent inhibitors than the parent compound.     

N-Acyl pyrazoles: Effective and tunable inhibitors of serine hydrolases

A series of N-acyl pyrazoles was examined as candidate serine hydrolase inhibitors in which the active site acylating reactivity and the leaving group ability of the pyrazole could be tuned not only through the nature of the acyl group (reactivity: amide?>?carbamate?>?urea), but also through pyrazole C4 substitution with electron-withdrawing or electron-donating substituents. Their impact on enzyme inhibitory activity displayed pronounced effects with the activity improving substantially as one alters both the nature of the reacting carbonyl group (urea?>?carbamate?>?amide) and the pyrazole C4 substituent (CN?>?H?>?Me). It was further demonstrated that the acyl chain of the N-acyl pyrazole ureas can be used to tailor the potency and selectivity of the inhibitor class to a targeted serine hydrolase. Thus, elaboration of the acyl chain of pyrazole-based ureas provided remarkably potent, irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent K_i?=?100–200?pM), dual inhibitors of FAAH and monoacylglycerol hydrolase (MGLL), or selective inhibitors of MGLL (IC₅₀?=?10–20?nM) while simultaneously minimizing off-target activity (e.g., ABHD6 and KIAA1363).     

Discovery of potent,highly selective covalent irreversible BTK inhibitors from a fragment hit

Bruton's Tyrosine Kinase (BTK) is a member of the TEC kinase family that is expressed in cells of hematopoietic lineage (e.g., in B cells, macrophages, monocytes, and mast cells). Small molecule covalent irreversible BTK inhibitor targeting Cys481 within the ATP-binding pocket, for example ibrutinib, has been applied in the treatment of B-cell malignancies. Starting from a fragment hit, we discovered a novel series of potent covalent irreversible BTK inhibitors that occupy selectivity pocket of the active site of the BTK kinase domain. Guided by X-ray structures and a fragment-based drug design (FBDD) approach, we generated molecules showing comparable cellular potency to ibrutinib and higher kinome selectivity against undesirable off-targets like EGFR.     

The Macamide N-3-Methoxybenzyl-Linoleamide Is a Time-Dependent Fatty Acid Amide Hydrolase (FAAH) Inhibitor

The Peruvian plant Lepidium meyenii (Maca) has been shown to possess neuroprotective activity both in vitro and in vivo [1–3]. Previous studies have also demonstrated the activity of the pentane extract and its macamides, the most representative lipophilic constituents of Maca, in the endocannabinoid system as fatty acid amide hydrolase (FAAH) inhibitors. One of the most active macamides, N-3-methoxybenzyl-linoleamide [4, 5], was studied to determine its mechanism of interaction with FAAH and whether it has inhibitory activity on mono-acyl glycerol lipase (MAGL), the second enzyme responsible for endocannabinoid degradation [6]. Macamide concentrations from 1 to 100 μM were tested using FAAH and MAGL inhibitor assay methods and showed no effect on MAGL. Tests with other conditions were performed in order to characterize the inhibitory mechanism of FAAH inhibition. N-3-methoxybenzyl-linoleamide displayed significant time-dependent and dose-dependent FAAH inhibitory activity. The mechanism of inhibition was most likely irreversible or slowly reversible. These results suggest the potential application of macamides isolated from Maca as FAAH inhibitors, as they might act on the central nervous system to provide analgesic, anti-inflammatory, or neuroprotective effects, by modulating the release of neurotransmitters.     

Piperidine and piperazine inhibitors of fatty acid amide hydrolase targeting excitotoxic pathology

FAAH inhibitors offer safety advantages by augmenting the anandamide levels “on demand” to promote neuroprotective mechanisms without the adverse psychotropic effects usually seen with direct and chronic activation of the CB1 receptor. FAAH is an enzyme implicated in the hydrolysis of the endocannabinoid N-arachidonoylethanolamine (AEA), which is a partial agonist of the CB1 receptor. Herein, we report the discovery of a new series of highly potent and selective carbamate FAAH inhibitors and their evaluation for neuroprotection. The new inhibitors showed potent nanomolar inhibitory activity against human recombinant and purified rat FAAH, were selective (>1000-fold) against serine hydrolases MGL and ABHD6 and lacked any affinity for the cannabinoid receptors CB1 and CB2. Evaluation of FAAH inhibitors 9 and 31 using the in vitro competitive activity-based protein profiling (ABPP) assay confirmed that both inhibitors were highly selective for FAAH in the brain, since none of the other FP-reactive serine hydrolases in this tissue were inhibited by these agents. Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on known FAAH cocrystal structures. To rationally design new molecules that are irreversibly bound to FAAH, we have constructed “precovalent” FAAH-ligand complexes to identify good binding geometries of the ligands within the binding pocket of FAAH and then calculated covalent docking poses to select compounds for synthesis. FAAH inhibitors 9 and 31 were evaluated for neuroprotection in rat hippocampal slice cultures. In the brain tissue, both inhibitors displayed protection against synaptic deterioration produced by kainic acid-induced excitotoxicity. Thus, the resultant compounds produced through rational design are providing early leads for developing therapeutics against seizure-related damage associated with a variety of disorders.     

Development and characterization of endocannabinoid hydrolases FAAH and MAGL inhibitors bearing a benzotriazol-1-yl carboxamide scaffold

A series of (1H-benzo[d][1,2,3]triazol-1-yl)(4-benzylpiperazin-1-yl)methanones and of (1H-benzo[d][1,2,3]triazol-1-yl)(4-phenylpiperazin-1-yl)methanones has been prepared and tested on human fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). In the benzylpiperazinyl series, compound 29 (ML30) exhibited an IC₅₀ value of 0.54 nM on MAGL, combined with a 1000-fold selectivity versus FAAH, while compounds 11 and 16 acted as potent dual FAAH-MAGL inhibitors (IC₅₀ <10 nM). In the phenylpiperazinyl series, compounds 37, 38, 42, and 43 displayed IC₅₀ values against MAGL in the nanomolar range, whilst being between one and two orders of magnitude less potent on the FAAH, while compounds 31 and 32 were potent FAAH inhibitors (IC₅₀ <20 nM) and over 12-fold selective versus MAGL. The key structural determinants driving the structure–activity relationships were explored by the minimization of the inhibitors inside the active site of both enzymes.     

Structure based design of novel irreversible FAAH inhibitors

Fatty acid amide hydrolase (FAAH) has attracted significant attention due to its promise as an analgesic target. This has resulted in the discovery of numerous chemical classes as inhibitors of this potential therapeutic target. In this paper we disclose a new series of novel FAAH irreversible azetidine urea inhibitors. In general these compounds illustrate potent activity against the rat FAAH enzyme. Our SAR studies allowed us to optimize this series resulting in the identification of compounds 13 which were potent inhibitors of both human and rat enzyme. This series of compounds illustrated good hydrolase selectivity along with good PK properties.     

Kinome-wide selectivity profiling of ATP-competitive mammalian target of rapamycin (mTOR) inhibitors and characterization of their binding kinetics

An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ～20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC(50) = 2 nM) relative to other inhibitors. In vitro biochemical profiling at 10 μM revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μM but did show that PP242 efficiently inhibited the RET receptor (EC(50), 42 nM) and JAK1/2/3 kinases (EC(50), 780 nM). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μM and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.     

A structure-activity relationship study on N-arachidonoyl-amino acids as possible endogenous inhibitors of fatty acid amide hydrolase

N-arachidonoyl-glycine (NAGly) has been recently identified in rodent tissues and found to exhibit analgesic activity in vivo. NAGly is a potent inhibitor of the fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for the degradation of the endocannabinoid N-arachidonoyl-ethanolamine (anandamide), and was shown recently to elevate the blood levels of the this analgesic compound. We have synthesized several N-arachidonoyl-amino acids of potential natural occurrence, as well as the D- and L-isomers of N-arachidonoyl-alanine, and have tested their activity on FAAH preparations from mouse, rat, and human cell lines, and from mouse or rat brain. The results indicate that the relative potency and enantioselectivity of N-arachidonoyl-amino acids as FAAH inhibitors depend on the animal species. Thus, whilst NAGly is the most potent compound on the rat and mouse enzymes, N-arachidonoyl-isoleucine is active only on human FAAH and N-arachidonoyl-alanine enantiomers show a varying degree of potency. Taken together, these data support the view that an enhancement of endogenous anandamide levels underlies in part the analgesic effects of NAGly in rodents.     

Anandamide and 2-arachidonoylglycerol inhibit fatty acid amide hydrolase by activating the lipoxygenase pathway of the arachidonate cascade

Treatment of intact human neuroblastoma CHP100 cells with anandamide (arachidonoylethanolamide, AEA) or 2-arachidonoylglycerol (2-AG) inhibits intracellular fatty acid amide hydrolase (FAAH). This effect was not associated with covalent modifications of FAAH, since specific inhibitors of farnesyltransferase, kinases, phosphatases, glycosyltransferase or nitric oxide synthase were ineffective. Electrophoretic analysis of (33)P-labelled proteins, Western blot with anti-phosphotyrosine antibodies, and glycan analysis of cellular proteins confirmed the absence of covalent modifications of FAAH. The inhibition by AEA was paralleled by an increased arachidonate release, which was not observed upon treatment of cells with linoleoylethanolamide, palmitoylethanolamide, or oleoylethanolamide. Moreover, cell treatment with AEA or 2-AG increased the activity of cyclooxygenase and 5-lipoxygenase, and the hydro(pero)xides generated from arachidonate by lipoxygenase were shown to inhibit FAAH, with inhibition constants in the low micromolar range. Consistently, inhibitors of 5-lipoxygenase, but not those of cyclooxygenase, significantly counteracted the inhibition of FAAH by AEA or 2-AG.     

Anandamide uptake is consistent with rate-limited diffusion and is regulated by the degree of its hydrolysis by fatty acid amide hydrolase

The uptake of arachidonoyl ethanolamide (anandamide, AEA) in rat basophilic leukemia cells (RBL-2H3) has been proposed to occur via a saturable transporter that is blocked by specific inhibitors. Measuring uptake at 25 s, when fatty acid amide hydrolase (FAAH) does not appreciably affect uptake, AEA accumulated via a nonsaturable mechanism at 37 degrees C. Interestingly, saturation was observed when uptake was plotted using unbound AEA at 37 degrees C. Such apparent saturation can be explained by rate-limited delivery of AEA through an unstirred water layer surrounding the cells (1). In support of this, we observed kinetics consistent with rate-limited diffusion at 0 degrees C. Novel transport inhibitors have been synthesized that are either weak FAAH inhibitors or do not inhibit FAAH in vitro (e.g. UCM707, OMDM2, and AM1172). In the current study, none of these purported AEA transporter inhibitors affected uptake at 25 s. Longer incubation times illuminate downstream events that drive AEA uptake. Unlike the situation at 25 s, the efficacy of these inhibitors was unmasked at 5 min with appreciable inhibition of AEA accumulation correlating with partial inhibition of AEA hydrolysis. The uptake and hydrolysis profiles observed with UCM707, VDM11, OMDM2, and AM1172 mirrored two selective and potent FAAH inhibitors CAY10400 and URB597 (at low concentrations), indicating that weak inhibition of FAAH can have a pronounced effect upon AEA uptake. At 5 min, the putative transport inhibitors did not reduce AEA uptake in FAAH chemical knock-out cells. This strongly suggests that the target of UCM707, VDM11, OMDM2, and AM1172 is not a transporter at the plasma membrane but rather FAAH, or an uncharacterized intracellular component that delivers AEA to FAAH. This system is therefore unique among neuro/immune modulators because AEA, an uncharged hydrophobic molecule, diffuses into cells and partial inhibition of FAAH has a pronounced effect upon its uptake.     

Design and synthesis of sulfoximine based inhibitors for HIV-1 protease

A new class of potent sulfoximine inhibitors for HIV-1 protease has been designed and synthesized. Substitution of the sulfoximine moiety into different parent compounds yields different inhibition effects. While our previously studied sulfoximine-based inhibitors display potency of 2.5 nM (IC(50)) against HIV-1 protease, introduction of the sulfoximine moiety into the asymmetric Indinavir yielded only micromolar inhibition. Docking studies showed structural variations in their modes of binding which explains this unexpected observation. The implication of these observations in the development of other sulfoximine inhibitors is discussed.     

Synthesis and evaluation of analogues of estrone-3-O-sulfamate as potent steroid sulfatase inhibitors

Estrone sulfamate (EMATE) is a potent irreversible inhibitor of steroid sulfatase (STS). In order to further expand SAR, the compound was substituted at the 2- and/or 4-positions and its 17-carbonyl group was also removed. The following general order of potency against STS in two in vitro systems is observed for the derivatives: The 4-NO(2) > 2-halogens, 2-cyano > EMATE (unsubstituted)>17-deoxyEMATE > 2-NO(2) > 4-bromo>2-(2-propenyl), 2-n-propyl > 4-(2-propenyl), 4-n-propyl > 2,4-(2-propenyl)= 2,4-di-n-propyl. There is a clear advantage in potency to place an electron-withdrawing substituent on the A-ring with halogens preferred at the 2-position, but nitro at the 4-position. Substitution with 2-propenyl or n-propyl at the 2- and/or 4-position of EMATE, and also removal of the 17-carbonyl group are detrimental to potency. Three cyclic sulfamates designed are not STS inhibitors. This further confirms that a free or N-unsubstituted sulfamate group (H(2)NSO(2)O-) is a prerequisite for potent and irreversible inhibition of STS as shown by inhibitors like EMATE and Irosustat. The most potent derivative synthesized is 4-nitroEMATE (2), whose IC(50)s in placental microsomes and MCF-7 cells are respectively 0.8 nM and 0.01 nM.     

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

Background

Combined 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 Findings

FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH^T488A-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/Significance

The 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.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-1-heteroalkyl-4-hydroxyquinolon-3-yl-benzothiadiazines

N-1-Alkylamino and N-1-alkyloxy-4-hydroxyquinolon-3-yl benzothiadiazines were synthesized and evaluated as inhibitors of genotype 1 HCV polymerase. The N-1-alkyloxy derivatives were not potent inhibitors, however N-1-alkylamino derivatives displayed comparable potency to carbon analogs. Analogs with aliphatic substituents were significantly more potent than those with benzylic substituents against genotype 1a polymerase. The most potent inhibitors contained small alkyl or carbocyclic substituents and exhibited IC50's of 50-100 and 200-400 nM against genotype 1b and 1a HCV polymerase, respectively.     

Structure–activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A2α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA₂α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA₂α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA₂α and FAAH, respectively.     

High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker

Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [³H]AEA, we have developed a human leukocyte assay using stably labeled [²H₄]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([²H₄]EA) was analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in the positive electrospray ionization (ESI) mode. The response for [²H₄]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [²H₄]AEA and HPLC–MS/MS method agreed well with those obtained using the [³H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC–MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials.