To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.

Fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. Inhibitors of FAAH are considered a potential therapeutic approach for the treatment of several nervous system disorders, including pain, anxiety, and insomnia. However, for FAAH inhibitors to achieve clinical utility, they must not only display efficacy in vivo but also selectivity for this enzyme relative to the numerous other serine hydrolases present in mammalian proteomes. Here, we report a general strategy for evaluating the pharmacological activity and target specificity of FAAH inhibitors and its implementation to develop the first class of selective reversible inhibitors of this enzyme that are highly efficacious in vivo. Using a series of functional proteomics, analytical chemistry, and behavioral pharmacology assays, we have identified a class of ␣-keto-heterocycles that show unprecedented selectivity for FAAH relative to other mammalian hydrolases, and, when administered to rodents, raise central nervous system levels of anandamide and promote cannabinoid receptor 1-dependent analgesia in several assays of pain sensation. These studies provide further evidence that FAAH may represent an attractive therapeutic target and describe a general route by which inhibitors of this enzyme can be optimized to achieve exceptional potency, selectivity, and efficacy in vivo.

Fatty acid amide hydrolase (FAAH) degrades neuromodulating fatty acid amides including anandamide (endogenous cannabinoid agonist) and oleamide (sleep-inducing lipid) at their sites of action and is intimately involved in their regulation. Herein we report the discovery of a potent, selective, and efficacious class of reversible FAAH inhibitors that produce analgesia in animal models validating a new therapeutic target for pain intervention. Key to the useful inhibitor discovery was the routine implementation of a proteomics-wide selectivity screen against the serine hydrolase superfamily ensuring selectivity for FAAH coupled with systematic in vivo examinations of candidate inhibitors.

Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP beta12-beta13 active site loop accounting for PP2A/4 potency and selectivity.

A systematic study of the structure-activity relationships (SAR) of 2b (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed targeting the C2 acyl side chain. A series of aryl replacements or substituents for the terminal phenyl group provided effective inhibitors (e.g., 5c, aryl = 1-napthyl, K i = 2.6 nM) with 5hh (aryl = 3-Cl-Ph, K i = 900 pM) being 5-fold more potent than 2b. Conformationally-restricted C2 side chains were examined and many provided exceptionally potent inhibitors of which 11j (ethylbiphenyl side chain) was established to be a 750 pM inhibitor. A systematic series of heteroatoms (O, NMe, S), electron-withdrawing groups (SO, SO 2 ), and amides positioned within and hydroxyl substitutions on the linking side chain were investigated which typically led to a loss in potency. The most tolerant positions provided effective inhibitors (12p, 6-position S, K i = 3 nM or 13d, 2-position OH, K i = 8 nM) comparable in potency to 2b. Proteomicwide screening of selected inhibitors from the systematic series of >100 candidates prepared revealed that they are selective for FAAH over all other mammalian serine proteases.The enzyme fatty acid amide hydrolase (FAAH) is the primary catabolic regulator of several bioactive lipid amides in vivo, including anandamide (1a) and oleamide (1b). 1-4 The central nervous system distribution of FAAH suggests that it degrades neuromodulating fatty acid amides at their sites of action and is intimately involved in their regulation. 5 Fatty acid amide hydrolase is currently the only characterized mammalian enzyme that is in the amidase signature family bearing an unusual catalytic Ser-Ser-Lys triad. 1,4,6-8 Recently, the crystal structure of FAAH cocrystallized with an irreversibly-bound arachidonyl fluorophosphonate confirmed its unusual catalytic triad and provided structural details of this enzyme. 1 Both anandamide (1a) 9 and oleamide (1b) 10-12 have emerged as prototypical members of the class of bioactive lipid amides 13,14 that serve as chemical messengers (Figure 1). Anandamide (1a), the most recognized member of the endogenous fatty acid ethanolamides, 15 binds and activates both the central type-1 (CB1) and peripheral type-2 (CB2) cannabinoid receptors. Anandamide (1a), and members of the cannabinoid family, 16 have been implicated in the modulation of nociception, 17-19 feeding, 20,21 emesis, anxiety, 22 cell proliferation, 23,24 inflammation, 25 memory 26 and neuroprotection after brain injury. 27 Thus, the Due to the potentially exciting therapeutic potential of inhibiting FAAH, there has been increasing interest in the development of potent inhibitors (Figure 2). 22,45-60 These include the discovery that the endogenous sleep-inducing molecule 2-octyl α-bromoacetoacetate is an effective FAAH inhibitor, 55 a series of reversible inhibitors bearing an electrophilic ketone 46,54,56 (e.g., trifluoromethyl ketone-based) that have not proven selective for FAAH over other mammalian serine hydrolases 61 and a set of irreversible inhibitor...

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The concurrent implementation of a proteome-wide serine hydrolase selectivity screen with traditional efforts to optimize fatty acid amide hydrolase inhibition potency leads to the expedited discovery of a new class of exceptionally potent and unusually selective inhibitors (I) with a 1,3,4-oxadiazole skeleton. The synthesis is outlined and the screening procedure is described. -(LEUNG, D.; DU, W.; HARDOUIN, C.; CHENG, H.; HWANG, I.; CRAVATT, B. F.; BOGER*, D. L.; Bioorg. Med. Chem. Lett. 15 (2005) 5,

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