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

Abstract

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH–URB597 complex at 2.3 Å resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 Å) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases.

Introduction

Fatty acid amide hydrolase (FAAH) degrades lipid signaling molecules such as the endogenous cannabinoid (endocannabinoid), anandamide (N-arachidonyl ethanolamine), and the sleep-inducing substance, oleamide (cis-9-octadecenamide).1, 2, 3 Pharmacological studies in rodents and experiments with knock-out mice have demonstrated that a blockade of FAAH activity raises endogenous levels of fatty acid amides.2, 3, 4, 5, 6 Such studies have also linked elevations in fatty acid amides to therapeutically desirable analgesic, anti-inflammatory, and neuropsychiatric effects.3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 These effects occur without many of the untoward side effects observed with direct cannabinoid receptor-1 agonists, such as hypothermia and cognitive and motor dysfunction. Finding selective and potent inhibitors of FAAH with drug-like properties has thus been the subject of intense medicinal chemistry efforts.17, 18 Many of the most promising inhibitors identified through these efforts are mechanism-based covalent modifiers of the enzyme.

FAAH is a member of the amidase signature family.1, 19, 20 Enzymes in this family are found in bacteria, archaea, and eukaryotes and hydrolyze diverse substrates ranging from glutamine²¹ to peptide amides²² to simple hydrophilic amides, such as acetamide or malonamide.²³ As might be expected, these enzymes have correspondingly diverse substrate binding pockets.20, 22, 23 In FAAH, a pocket adjacent to the catalytic site branches into an acyl-chain binding pocket and a tunnel leading to the membrane (membrane access channel)²⁰ (Fig. 1a). Consistent with their proposed roles in binding the fatty acid chains of substrates, these portions of FAAH have hydrophobic surfaces.²⁰ A second channel, the cytosolic port, connects the active site to the cytoplasm. This hydrophilic cavity interacts with the head groups of FAAH substrates.20, 24, 25 It provides a route through which hydrophilic products (ethanolamine in the case of anandamide) and the leaving groups of certain covalent inhibitors are released into the cytoplasm.20, 24, 25 FAAH and its homolog FAAH-2²⁶ are unusual members of the amidase signature family in that they are membrane associated. Nevertheless, FAAH shares a core set of residues, including a catalytic Ser-Ser-Lys triad, with other members of the family.20, 22, 23

The catalytic mechanism of FAAH has attracted considerable interest.2, 27, 28, 29 In broad outline, it involves four steps: (1) nucleophilic attack of the substrate by an activated serine to generate an enzyme-linked tetrahedral intermediate, (2) subsequent elimination of a substrate leaving group to form an acyl–enzyme intermediate, (3) attack of an activated water to create a second tetrahedral intermediate, and (4) resolution into the acid product and regenerated enzyme (Fig. 1b). Ser241 was identified as the nucleophile in the active site of FAAH through mutational and affinity labeling studies.²⁷ Mutational studies also showed the importance of Lys142, which acts as both a base to activate Ser241 and as an acid that helps protonate the substrate leaving group.²⁸ Structural studies established that both of these activities of Lys142 are indirect and mediated through the hydroxyl side chain of Ser217.²⁰ FAAH is an atypical serine hydrolase; it catalyzes the hydrolysis of both amide and ester substrates at comparable rates, with an acylation rate-limiting step.²⁸ It has been proposed that this unusual enzymatic profile is critical for FAAH activity in vivo, where lipid esters are far more abundant than lipid amides.²⁸ Despite these differences, some classes of FAAH inhibitors, such as carbamates, fluorophosphonates, and α-ketoheterocycles, also inhibit non-homologous serine hydrolases, suggesting that the enzymes share some degree of mechanistic similarity.2, 17

URB597 is a widely used carbamate inhibitor of FAAH with a relatively simple chemical structure, consisting of a cyclohexyl moiety, a carbamate reactive group, and a meta-biphenylamide leaving group (Fig. 1c).30, 31 It displays excellent selectivity for FAAH in the nervous system, although the inhibitor does inactivate additional peripheral hydrolases.³² The carbamate inhibitors were initially derived from an acetylcholine esterase inhibitor with very poor FAAH potency (compound 19, Fig. 1c) but were rapidly optimized into compounds such as URB524, which has an apparent potency of 63 nM (Fig. 1c).³⁰ Addition of an amide group to the meta-biphenyl moiety yielded URB597, an inhibitor with an IC₅₀ of 4.6 nM,³¹ making it one of the earliest drug-like inhibitors of FAAH. Initial modeling studies suggested that the biphenyl moiety of the URB compounds might bind in the hydrophobic pocket of FAAH, with the cyclohexyl group extending into the cytosolic port.30, 31 Subsequent experiments indicated that the original modeling was incorrect, instead invoking a flipped orientation of the compounds in the active site of FAAH.³³

Here, we describe the URB597-carbamoylated structure of humanized rat FAAH (h/rFAAH), determined at 2.3 Å resolution. The h/rFAAH protein used in these and past crystallographic studies contains six active-site mutations that replace non-conserved residues in the rat sequence with their human counterparts.²⁵ These mutations allow us to take advantage of the superior expression and biophysical properties of rat FAAH while mimicking the inhibitor binding profile of the human enzyme. Because it contains a plateau composed of two helices with exposed hydrophobic residues, h/rFAAH remains fully membrane-associated even though it lacks the hydrophobic N-terminal sequence of the wild-type enzyme.²⁰ The structure of FAAH–URB597 establishes the orientation of the cyclohexyl-carbamate moiety within the acyl-chain binding pocket and is consistent with the results of structure–activity relationship experiments. The structure also shows the location of a water molecule in the active site that is close to the carbonyl carbon of the inhibitor. The water is held in place through hydrogen bonds to both protein and a second water molecule. Re-determination of the structure of the urea-based inhibitor, PF-3845,¹⁸ at a higher resolution reveals similarly placed waters. The water molecules close to the inhibitor carbonyl carbon atom in the active site of FAAH superimpose on those seen in high-resolution structures of non-homologous serine hydrolases, indicating that some of the mechanistic insights derived from decades of analysis of serine proteases may be applicable to FAAH.