Structure-guided inhibitor design for human FAAH by interspecies active site conversion

Abstract

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-Å crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.anandamide ͉ crystal structure ͉ endocannabinoid ͉ fatty acid amides ͉ hydrolase F atty acid amide hydrolase (FAAH) is an integral membrane enzyme that hydrolyzes the fatty acid amide class of lipid transmitters (1, 2). FAAH substrates include the endogenous cannabinoid N-arachidonoyl ethanolamine (anandamide) (3), the antiinflammatory factor N-palmitoyl ethanolamine (PEA) (4), the sleep-inducing substance 9(Z)-octadecenamide (oleamide) (5), and the satiating signal N-oleoyl ethanolamine (OEA) (6). FAAH inactivation by either chemical inhibition or genetic deletion of the FAAH gene leads to elevated endogenous levels of fatty acid amides and a range of behavioral effects that include analgesia (7-12), anxiolytic (8,13,14), antidepressant (13, 15), sleep-enhancing (16), and antiinflammatory (17-19) phenotypes. Importantly, these behavioral phenotypes occur in the absence of alterations in motility, weight gain, or body temperature that are typically observed with direct cannabinoid receptor 1 (CB1) agonists. Inhibition of FAAH thus may offer an attractive way to produce the therapeutically beneficial phenotypes of activating the endocannabinoid system without the undesirable side effects that are observed with direct CB1 agonists.FAAH is a member of a large class of enzymes termed the amidase signature class (20). These enzymes, which span all kingdoms of life, use an unusual Ser-Ser-Lys catalytic triad (21, 22) to hydrolyze amide bonds on a wide range of small-molecule substrates. Despite their atypical catalytic mechanism, amidase signature enzymes are inactivated by general classes of serine hydrolase inhibitors [e.g., trifluoromethyl ketones (23, 24), fluorophosphonates (25), ␣-ketoheterocycles (26), carbamates (8, 27)]. First-generation FAAH inhibitors, such as methyl arachidonyl fluorophosphonate (MAFP) (25), were substrate-derived in structure and therefore lack selectivity for FAAH relative to other l...

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

“…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.…”

“…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.…”

Chiral 3-(4,5-dihydrooxazol-2-yl)phenyl alkylcarbamates as novel FAAH inhibitors: Insight into FAAH enantioselectivity by molecular docking and interaction fields

“…In particular, in this approach, we focused on exploring the differences arising from the protein-ligand interactions of the recognition process, as it can be assumed that the reactivity of the compounds is in similar level within the enantiomeric pairs. It should be noted that a crystal structure of partially humanized rat FAAH with a druglike inhibitor PF-750 [41] (PDB code 2VYA) has been published recently. This structure shows structural rearrangements in the substrate access channel region with Phe432 flipping into the acyl chain binding (ACB) channel.…”

Preclinical Characterization of the FAAH Inhibitor JNJ-42165279

“…30 Urea derivatives as prepared by Pfizer, Takeda, and us operate via a similar mechanism but have aromatic amines as leaving groups rather than alcohols or phenols. 31 40 Amgen, 41 Renovis, 42 and Johnson and Johnson. 43 Several compounds have been profiled to some degree in the clinic.…”