Metabolic Serine Hydrolase Research Articles

The Metabolic Serine Hydrolases and Their Functions in Mammalian Physiology and Disease

The serine hydrolase (SH) superfamily consists of >200 enzymes in humans characterized by the presence of an active site serine that is used for the hydrolysis of substrates. The membership of this enzyme class is near-equally split between serine proteases (trypsin/chymotrypsin/subtilisin enzymes) and ‘metabolic’ SHs that cleave ester, amide, or thioester bonds in small molecules, peptides, or proteins.1 The serine proteases have been the subject of several books and reviews.2–5 This article will instead focus on the metabolic SHs (Fig. 1). Fig. 1 Dendrogram showing the primary sequence alignment of the human mammalian metabolic SHs, where alignment was generated by anchoring sequences at the site of their catalytic Ser residues. The nucleophilicity of the active site serine of metabolic SHs arises from its participation in a catalytic dyad (e.g. Ser-Lys or Ser-Asp) or triad (e.g. Ser-His-Asp or Ser-Ser-Lys).6,7 The SH catalytic mechanism proceeds by formation of an acyl-enzyme intermediate at the active site serine, followed by water-induced saponification of the product, and regeneration of the free serine residue for entry into the next reaction cycle (Fig. 2).8,9 Owing to the enhanced reactivity of the active site serine, the functional state of most SHs can be assessed using active-site directed affinity labels such as fluorophosphonates (FPs, Fig. 2).1,10 Fig. 2 (A) Mechanism of SH catalysis. (B) Mechanism of SH labeling by the active site-directed activity-based probe fluorophosphonate-biotin (FP-biotin). (C) Three dimensional structure of MGLL, a SH with a canonical α/β-hydrolase fold. The serine nucleophile of metabolic SHs is generally, though not exclusively embedded within a GXSXG motif and a majority these enzymes adopt an α/β hydrolase fold that consists of a central β-sheet surrounded by α-helicies (Fig. 2).11 This superfamily also encompasses other smaller subsets of structurally distinct enzymes such as the phospholipase A2s, the amidase signature enzymes, and the dipeptidylpeptidases.12,13 Metabolic SHs have been shown to participate in virtually all (patho)physiological processes in mammals, including neurotransmission,14 metabolism,15 pain sensation,16 inflammation,17 oxidative stress,18 cancer,19 and bacterial infection.20 Many excellent reviews have described the structure and function of individual SHs.15,19,21–23 Here, we attempt to provide a comprehensive summary that captures our state of knowledge about mammalian metabolic SHs in their entirety, including those enzymes that remain mostly or completely uncharacterized. Particular emphasis will be placed on relating the biochemistry and enzymology of individual SHs to the physiological substrates and products that they regulate in living systems, and how SHs, through the regulation of specific metabolic pathways impact health and disease. If selective and efficacious inhibitors are available for a particular SH, we will also include a discussion of their use. The majority of this review will be organized by substrate class. Later, we will discuss SHs for which putative endogenous substrates have not been identified, as well as emerging chemoproteomic and metabolomic methods aimed at assigning functions to these enzymes. For the sake of consistency, we have elected to refer to SHs by their proper gene names throughout this review (rather than their common name or abbreviation), but have also attempted to include other aliases if possible.

Novel Mechanistic Class of Fatty Acid Amide Hydrolase Inhibitors with Remarkable Selectivity

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that degrades the fatty acid amide family of signaling lipids, including the endocannabinoid anandamide. Genetic or pharmacological inactivation of FAAH leads to analgesic, anti-inflammatory, anxiolytic, and antidepressant phenotypes in rodents without showing the undesirable side effects observed with direct cannabinoid receptor agonists, indicating that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system disorders. However, the FAAH inhibitors reported to date lack drug-like pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750) and N-phenyl-4-(quinolin-2-ylmethyl)piperazine-1-carboxamide (PF-622) as a novel mechanistic class of FAAH inhibitors. PF-750 and PF-622 show higher in vitro potencies than previously established classes of FAAH inhibitors. Rather unexpectedly based on the high chemical stability of the urea functional group, PF-750 and PF-622 were found to inhibit FAAH in a time-dependent manner by covalently modifying the enzyme's active site serine nucleophile. Activity-based proteomic profiling revealed that PF-750 and PF-622 were completely selective for FAAH relative to other mammalian serine hydrolases. We hypothesize that this remarkable specificity derives, at least in part, from FAAH's special ability to function as a C(O)-N bond hydrolase, which distinguishes it from the vast majority of metabolic serine hydrolases in mammals that are restricted to hydrolyzing esters and/or thioesters. The piperidine/piperazine urea may thus represent a privileged chemical scaffold for the synthesis of FAAH inhibitors that display an unprecedented combination of potency and selectivity for use as potential analgesic and anxiolytic/antidepressant agents.