Sirtuins catalyze NAD؉ -dependent protein deacetylation and are critical regulators of transcription, apoptosis, metabolism, and aging. There are seven human sirtuins (SIRT1-7), and SIRT1 has been implicated as a key mediator of the pathways downstream of calorie restriction that have been shown to delay the onset and reduce the incidence of age-related diseases such as type 2 diabetes. Increasing SIRT1 activity, either by transgenic overexpression of the Sirt1 gene in mice or by pharmacological activation by small molecule activators resveratrol and SRT1720, has shown beneficial effects in rodent models of type 2 diabetes, indicating that SIRT1 may represent an attractive therapeutic target. Herein, we have assessed purported SIRT1 activators by employing biochemical assays utilizing native substrates, including a p53-derived peptide substrate lacking a fluorophore as well as the purified native full-length protein substrates p53 and acetyl-CoA synthetase1. SRT1720, its structurally related compounds SRT2183 and SRT1460, and resveratrol do not lead to apparent activation of SIRT1 with native peptide or full-length protein substrates, whereas they do activate SIRT1 with peptide substrate containing a covalently attached fluorophore. Employing NMR, surface plasmon resonance, and isothermal calorimetry techniques, we provide evidence that these compounds directly interact with fluorophorecontaining peptide substrates. Furthermore, we demonstrate that SRT1720 neither lowers plasma glucose nor improves mitochondrial capacity in mice fed a high fat diet. SRT1720, SRT2183, SRT1460, and resveratrol exhibit multiple off-target activities against receptors, enzymes, transporters, and ion channels. Taken together, we conclude that SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1.

IntroductionChemical signals, or neurotransmitters, represent the fundamental mode for intercellular communication in the nervous system. (1) The classical model for neurotransmitter action involves the uptake and storage of these small molecules into synaptic vesicles, release of vesicular contents into the synaptic cleft in response to depolarization of the presynaptic terminal by an action potential, binding of released neurotransmitters to cognate protein receptors on the postsynaptic (and presynaptic) terminal, and, finally, termination of signaling by protein-mediated uptake and degradation of neurotransmitters from the synaptic cleft. This model applies to a large number of well-studied neurotransmitters, including glutamate, γ-amino butyric acid (GABA), acetylcholine, and the monoamines, all of which represent aqueous solution-soluble molecules. More recently, lipids have emerged as an important class of chemical messengers in the nervous system that operate by a distinct mechanism.The hydrophobic nature of lipids precludes their stable uptake and storage into synaptic vesicles. Instead, lipid messengers appear to be biosynthesized and released by neurons at the moment of their intended action, which is often referred to as "on-demand" production. Similarly, the capacity of lipids to freely cross cell membranes places the burden of signal termination largely on the action of degradative enzymes. Lipid signaling systems are thus embedded within an elaborate collection of metabolic pathways, the composition and regulation of which ultimately establish the magnitude and duration of transmitter action. Here, we will review these general concepts as they relate to a specific class of lipid transmitters, the endogenous cannabinoids (endocannabinoids), and highlight how delineation of their cognate metabolic enzymes has been translated into the development of chemical and genetic tools to test the role that the endocannabinoid system plays in nervous system signaling and behavior.Endocannabinoids are defined as endogenous small molecules that activate the cannabinoid receptors CB1 and CB2, which are G-protein-coupled receptors that also recognize Δ 9tetrahydrocannabinol (THC), the psychoactive component of marijuana. (2, 3) The CB1 receptor is the major cannabinoid receptor in the nervous system and is responsible for mediating most of the neurobehavioral effects of THC. (4, 5) The CB2 receptor is predominantly expressed in immune cells, (6) where it appears to play a role in mediating the immunosuppressive effects of cannabinoids. Two principal endocannabinoids have been identified in mammals, N-arachidonoyl ethanolamine (anandamide) (7) and 2-Anandamide was the first identified endogenous ligand for the CB1 receptor. (7) As will be described in the following section, anandamide and other NAEs are produced upon demand through activity-dependent cleavage of membrane lipid precursors. The biological activity of anandamide in the central nervous system and in peripheral tissues is terminated by its removal from th...

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo as determined by activity-based protein profiling and raises brain anandamide levels for up to 24 hrs, resulting in profound cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.

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...