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 druglike pharmacokinetic properties and/or selectivity. Herein we describe piperidine/piperazine ureas represented by 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.Fatty acid amide hydrolase (FAAH 1 ) is an integral membrane enzyme (1, 2) that regulates the fatty acid amide family of lipid transmitters, which includes the endogenous cannabinoid N-arachidonyl ethanolamine (anandamide) (3), the anti-inflammatory 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 genetic deletion of the FAAH gene in mice (7) or by chemical inhibitors (8) leads to elevated endogenous levels of fatty acid amides with concomitant analgesic (9-13), anxiolytic (8), antidepressant (14, 15), sleep-enhancing (16), and anti-inflammatory (9,17,18) phenotypes. Notably the behavioral phenotypes observed in FAAH knockout (-/-) mice (7, 9) or in rodents treated with FAAH inhibitors (8,(11)(12)(13)(14)18) occur in the absence of alterations in motility, weight gain, or body temperature that are typically observed with direct cannabinoid receptor 1 (CB1) agonists. These findings suggest that FAAH may represent an attractive therapeutic target for treatment of pain, inflammation, and other central nervous system (CNS) diso...

Endothelins are peptide hormones with a potent vasoconstrictor activity that are also known to function as intercellular signaling molecules. The final step in the biosynthesis of endothelins is the proteolytic processing of precursor peptides by endothelin-converting enzymes (ECEs). ECE-1 is a zinc metalloendopeptidase related in amino acid sequence to neprilysin, a mammalian cell-surface peptidase involved in the metabolism of numerous biologically active peptides. Despite apparent structural similarities, ECE-1 and neprilysin have been considered to differ significantly in substrate specificity. In this study we have examined the activity of recombinant ECE-1 against a collection of biologically active peptides. ECE-1, unlike neprilysin, was found to have minimal activity against substrates smaller than hexapeptides, such as Leu-enkephalin. Larger peptides such as neurotensin, substance P, bradykinin, and the oxidized insulin B chain were hydrolyzed by ECE-1 as efficiently as big endothelin-1, a known in vivo substrate. Identification of the products of hydrolysis of six peptides indicates that ECE-1 has a substrate specificity similar to that of neprilysin, preferring to cleave substrates at the amino side of hydrophobic residues. The data indicate that ECE-1 possesses a surprisingly broad substrate specificity and is potentially involved in the metabolism of biologically active peptides distinct from the endothelins. Endothelins (ETs)1 are potent vasoconstrictive peptides of 21 amino acids produced by vascular endothelial cells (1). Three ET isoforms, ET-1, ET-2, and ET-3, encoded by distinct genes, are known to exist in humans (2). Endothelins are involved in the regulation of vascular tone and may also play roles in various cardiovascular and renal diseases (3). ETs are also required during embryonic development for the intercellular signaling necessary for the proper development of neural crestderived tissues (4). The final step in the biosynthesis of the endothelins is the conversion of 38 -41 residue precursors (big ETs) to the active hormones via the cleavage of a Trp 21 -Val/ Ile 22 bond by endothelin-converting enzymes (ECEs (5)). ECE-1 has been purified from vascular endothelium, endothelial cell lines, and lung microsomes (6 -8). ) is a Type II integral membrane protein expressed by endothelial cells in tissues such as aorta, lung, ovary, and testis. It has also been reported to be expressed by endocrine cells such as adrenal chromaffin cells and pancreatic ␤ cells (9). Targeted disruption of the ECE-1 gene has shown that ECE-1 is the physiologically relevant activating enzyme for both ET-1 and ET-3 in vivo (10).Molecular cloning of mammalian ECE-1 cDNAs has demonstrated the existence of three mRNAs transcribed from a single gene (11,12). The proteins encoded by these RNAs have identical catalytic domains but differ only in their NH 2 -terminal amino acid sequence. Two of the ECE-1 isoforms are expressed on the cell surface; the other is localized in the trans-Golgi network (12) An additional isof...

Strategies employing non-gel based methods for quantitative proteomic profiling such as isotope coded affinity tags coupled with mass spectrometry (ICAT-MS) are gaining attention as alternatives to two-dimensional gel electrophoresis (2-DE). We have conducted a large-scale investigation to determine the degree of reproducibility and depth of proteome coverage of a typical ICAT-MS experiment by measuring protein changes in Escherichia coli treated with triclosan, an inhibitor of fatty acid biosynthesis. The entire ICAT-MS experiment was conducted on four independent occasions where more than 24 000 peptides were quantitated using an ion-trap mass spectrometer. Our results demonstrated that quantitatively, the technique provided good reproducibility (median coefficient of variation of ratios was 18.6%), and on average identified more than 450 unique proteins per experiment. However, the method was strongly biased to detect acidic proteins (pI < 7), under-represented small proteins (<10 kDa) and failed to show clear superiority over 2-DE methods in monitoring hydrophobic proteins from cell lysates.

Matrix-assisted laser desorption ionization (MALDI) mass spectra have been obtained directly from thin-layer isoelectric focusing (IEF) gels with as little as 700 femtomoles of alpha- and beta-chain bovine hemoglobin and bovine carbonic anhydrase, and 2 picomoles of bovine trypsinogen, soybean trypsin inhibitor, and bovine serum albumin all loaded onto a single lane. By soaking the gel in a matrix solution, matrix was deposited over the entire gel surface, allowing MALDI scanning down complete lanes of the one-dimensional gel. As long as matrix crystals were deposited finely on the surface of the gel, time-lag focusing techniques were capable of ameliorating some of the mass accuracy limitations inherent in desorbing from uneven insulator surfaces with external calibration. Eleven measurements on the 5 kDa alpha-subunit proteins of lentil lectin measured over the course of 1 h and referenced to a single calibration yielded a standard deviation of 0.025%. Colloidal gold staining was found to be compatible with desorption directly from IEF and sodium dodecyl sulfate (SDS)-polyacrylamide gels. This direct approach simplifies the interface between gel electrophoresis and mass spectrometry dramatically, making the process more amenable to automation.