Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

Mileni, Mauro; Kamtekar, S.; Wood, David; Benson, Timothy E.; Cravatt, Benjamin F.; Stevens, Raymond C.

doi:10.1016/j.jmb.2010.05.034

Cited by 93 publications

(121 citation statements)

References 52 publications

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“…The overall structure of the FAAH protein resembles those previously reported (36)(37)(38)(39)(40)(41). The core structure of the FAAH monomer adopts an α/β fold with a twisted 11-strand β-sheet in the center and 24 α-helices surrounding the sheet ( Fig.…”

Section: Resultssupporting

confidence: 75%

“…These recently reported inhibitors are highly potent and selective, with some of them demonstrating in vivo efficacy (26,35). The inhibitor cocrystal structures were also reported for MAFP with rat FAAH, and PF-750, PF-3845, URB597, and OL-135 with humanized rat FAAH (36)(37)(38)(39)(40)(41). However, like ATMK and MAFP, most of the inhibitors disclosed so far rely on covalent modification of the catalytic serine 241 in the active site, either by forming a stable acyl-enzyme adduct or a hemi-acetal with Ser241, which is stabilized by the oxyanion hole.…”

Section: Discovery and Molecular Basis Of Potent Noncovalent Inhibitomentioning

confidence: 80%

“…To date, each of the disclosed FAAH structures is in its ligand-bound form; from the first rat FAAH structure in complex with a substrate-inspired irreversible arachidonyl inhibitor to the morerecent structures including human-rat chimeric FAAH in complex with various inhibitors (36)(37)(38)(39)(40)(41). While the main chain of the protein showed very little movement from one inhibitor complex to another, the side chains of a few residues lining the active site were found to be rather flexible.…”

Section: Discussionmentioning

confidence: 99%

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Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

Min¹,

Thibault²,

Porter

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Fatty acid amide hydrolase (FAAH), an amidase-signature family member, is an integral membrane enzyme that degrades lipid amides including the endogenous cannabinoid anandamide and the sleep-inducing molecule oleamide. Both genetic knock out and pharmacological administration of FAAH inhibitors in rodent models result in analgesic, anxiolytic, and antiinflammatory phenotypes. Targeting FAAH activity, therefore, presents a promising new therapeutic strategy for the treatment of pain and other neurological-related or inflammatory disorders. Nearly all FAAH inhibitors known to date attain their binding potency through a reversible or irreversible covalent modification of the nucleophile Ser241 in the unusual Ser-Ser-Lys catalytic triad. Here, we report the discovery and mechanism of action of a series of ketobenzimidazoles as unique and potent noncovalent FAAH inhibitors. Compound 2, a representative of these ketobenzimidazoles, was designed from a series of ureas that were identified from highthroughput screening. While urea compound 1 is characterized as an irreversible covalent inhibitor, the cocrystal structure of FAAH complexed with compound 2 reveals that these ketobenzimidazoles, though containing a carbonyl moiety, do not covalently modify Ser241. These inhibitors achieve potent inhibition of FAAH activity primarily from shape complementarity to the active site and through numerous hydrophobic interactions. These noncovalent compounds exhibit excellent selectivity and good pharmacokinetic properties. The discovery of this distinctive class of inhibitors opens a new avenue for modulating FAAH activity through nonmechanism-based inhibition.structure-based design | medicinal chemistry F atty acid amides (FAAs) represent a class of lipid signaling molecules that modulate a wide range of physiological processes in the central nervous system (1). As a prominent member of the FAA family, the endogenous cannabinoid anandamide, also known as N-arachidonoylethanolamine or AEA, produces its neurotransmitting effects by binding to and activating two G protein-coupled cannabinoid receptors (i.e., CB1 in the central nervous system and CB2 in the periphery) (2). Previous studies have shown that the effects of anandamide include neurological pain suppression, antiproliferation of cancer cells, enhancement of feeding behavior, and generation of motivation and pleasure (3-7). Endogenous oleamide (cis-9,10-octadecenoamide), also known as a sleep-inducing molecule in the FAA family, was found in the cerebrospinal fluid of sleep-deprived animals (8, 9). In addition, other neurological activities have been associated with oleamide, including regulation of memory processes, body temperature, and locomotor activity (10-12). However, the actions of these lipid messengers are ephemeral because of the hydrolytic activity of a number of enzymes including fatty acid amide hydrolase (FAAH). FAAH is an integral membrane enzyme that degrades these lipid amides (13-17) and thus terminates their activities. Inhibition of FAAH activity provi...

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Section: Resultssupporting

confidence: 75%

Section: Discovery and Molecular Basis Of Potent Noncovalent Inhibitomentioning

confidence: 80%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

Min¹,

Thibault²,

Porter

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Adjacent to the cyclohexyl group, the carbamate group was confirmed to form a covalent bond with Ser241 (Figures 15a and 15b). 74 The biphenyl moiety, which serves as a leaving group, was located in the CP.…”

Section: Carbamatesmentioning

confidence: 99%

Therapeutic Potential of Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase, and N-Acylethanolamine Acid Amidase Inhibitors

et al. 2016

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“…In the case of FAAH, ARN2508-mediated inhibition was attributed to a covalent bond formed between the carbamate moiety of the inhibitor and the enzyme's catalytic serine (14). Thus, the carbamate functional group of ARN is required for FAAH inhibition; however, it is not necessary for inhibition of COX.…”

mentioning

confidence: 99%

Dual cyclooxygenase–fatty acid amide hydrolase inhibitor exploits novel binding interactions in the cyclooxygenase active site

Goodman

Rouzer

et al. 2018

Journal of Biological Chemistry

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The cyclooxygenases COX-1 and COX-2 oxygenate arachidonic acid (AA) to prostaglandin H2 (PGH2). COX-2 also oxygenates the endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA), to the corresponding PGH2 analogs. Both enzymes are targets of nonsteroidal anti-inflammatory drugs (NSAIDs), but NSAID-mediated COX inhibition is associated with gastrointestinal toxicity. One potential strategy to counter this toxicity is to also inhibit fatty acid amide hydrolase (FAAH), which hydrolyzes bioactive fatty acid ethanolamides (FAEs) into fatty acids and ethanolamine. Here, we investigated the mechanism of COX inhibition by ARN2508, an NSAID that inhibits both COXs and FAAH with high potency, target selectivity, and decreased gastrointestinal toxicity in mouse models, presumably due to its ability to increase levels of FAEs. A 2.27 Å resolution X-ray crystal structure of the COX-2•(S)-ARN2508 complex reveals that ARN2508 adopts a binding pose similar to that of its parent NSAID flurbiprofen. However, ARN2508's alkyl tail is inserted deep into the top channel, an active site region not exploited by any previously reported NSAID. As for flurbiprofen, ARN2508's potency is highly dependent on the configuration of the -methyl group. Thus, (S)-ARN2508 is more potent than (R)-ARN2508 for inhibition of AA oxygenation by both COXs and 2-AG oxygenation by COX-2. Also, similarly to (R)-flurbiprofen, (R)-ARN2508 exhibits substrate-selectivity for inhibition of 2-AG oxygenation. Site-directed mutagenesis confirms the importance of insertion of the alkyl tail into the top channel for (S)-ARN2508's potency and suggests a role for Ser-530 as a determinant of the inhibitor's slow rate of inhibition compared to that of (S)-flurbiprofen.The enzyme cyclooxygenase (COX) catalyzes the committed step in prostanoid biosynthesis, the bis-dioxygenation and cyclization of AA to form the endoperoxy-hydroperoxide intermediate prostaglandin (PG)G2. Then, via its peroxidase activity, the enzyme reduces the 15-hydroperoxy group of PGG2 to produce the final product, PGH2 (1). PGH2 is a substrate for various terminal prostanoid synthases that catalyze the formation of COX-2•ARN2508 Structure and Inhibition Kinetics2 PGs, as well as thromboxane A2. PGs exert various biological effects including platelet aggregation, gastrointestinal and cardiovascular regulation, parturition, and modulation of the inflammatory response (2).There are two COX isoforms, COX-1, and COX-2, distinguished primarily by their patterns of expression. Specifically, the expression of COX-2 is induced in response to pro-inflammatory cytokines and growth factors, whereas the expression of COX-1 is constitutive in most tissues (3). In addition, due to its larger active site, COX-2 can oxygenate analogues of AA that are poor substrates for COX-1. These include the endocannabinoids, 2-arachidonoylglycerol (2-AG) and arachidonoylethanolamide (AEA), oxygenation of which yields PGH2-glyceryl ester and PGH2-ethanolamide (PGH2-EA), respectively (4-6).Both CO...

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Crystal Structure of Fatty Acid Amide Hydrolase Bound to the Carbamate Inhibitor URB597: Discovery of a Deacylating Water Molecule and Insight into Enzyme Inactivation

Cited by 93 publications

References 52 publications

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

Discovery and molecular basis of potent noncovalent inhibitors of fatty acid amide hydrolase (FAAH)

Therapeutic Potential of Fatty Acid Amide Hydrolase, Monoacylglycerol Lipase, and N-Acylethanolamine Acid Amidase Inhibitors

Dual cyclooxygenase–fatty acid amide hydrolase inhibitor exploits novel binding interactions in the cyclooxygenase active site

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