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.

ABSTRACTzheimer disease (AD) is characterized by deposits of an aggregated 42-amino-acid P-amyloid peptide (I3AP) in the brain and cerebrovasculature. After a concentration-dependent lag period during in vitro incubations, soluble preparations of synthetic .8AP slowly form fibrillar aggregates that resemble natural amyloid and are measurable by sedimentation and thioflavin T-based fluorescence. Aggregation of soluble flAP in these in vitro assays is enhanced by addition ofsmall amounts ofpre-aggregated gamyloid "seed" material. We also have prepared these seeds by using a naturally occurring reaction between glucose and protein amino groups resulting in the formation of advanced "glycosylation" end products (AGEs) which chemically crosslink proteins. AGE-modified flAP-nucleation seeds further accelerated aggregation of soluble flAP compared to non-modiflied "seed" material. To better understand the factors which contribute to amyloid formation and stability, we have studied the in vitro aggregation of soluble synthetically prepared PAP monomers, a process which displays nucleation-dependent kinetics, especially at physiological (nanomolar) concentrations of monomer (refs. 9 and 10; this report). Although millimolar concentrations of (3AP exhibit extensive aggregation within minutes, micromolar and lower concentrations of monomer display a concentration-dependent lag period during which little or no measurable aggregate is formed, followed by a "growth" phase of more rapid aggregation (9-11). This lag period can be eliminated by adding trace amounts of preformed aggregate as "seed" material which serves to immediately induce aggregation (9). Seeding apparently eliminates the need for de novo formation of aggregation nuclei, a much slower process than the subsequent growth of nucleated aggregates. Thus, amyloid seeds can be seen to represent critical protein masses with special structural features capable of nucleating the formation of larger insoluble aggregates of amyloid from pools of soluble P3AP. The nature of these structural characteristics is unknown, but the net result of seeding is significant acceleration in the rate of aggregation compared with unseeded incubations of soluble PAP. By extrapolation from this simple in vitro model to the similar nanomolar concentrations of soluble fAP found in cerebrospinal fluid in vivo, it might be expected that the deposition and accumulation of PAP as amyloid plaques would reflect, in part, the amount or availability of seed material able to nucleate aggregation. Some cases of AD might correspondingly arise as a consequence of increased availability of nucleation seeds in the disease-prone brain relative to normal counterparts not destined to suffer AD at a similar chronological age.In the present communication, we explore the possibility that the formation or stability of amyloid structures which seed PAP aggregation may be enhanced by covalent crosslinks that chemically polymerize components of the aggregate. Under physiological conditions, long-lived proteins beco...

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...

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1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) bioconjugations have been utilized in preparing variants for medical research. While there have been advances in optimizing the reaction for aqueous applications, there has been limited focus toward identifying conditions and side reactions that interfere with product formation. We present a systematic investigation of EDC/N-hydroxysulfosuccinimide (sNHS)-mediated bioconjugations on carboxylated peptides and small proteins. We identified yet-to-be-reported side products arising from both the reagents and substrates. Model peptides used in this study illustrate particular substrates are more susceptible to side reactions than others. From our studies, we found that bioconjugations are more efficient with high concentrations of amine nucleophile but not sNHS. Performing bioconjugations on a model affibody protein show that the trends established with model peptides hold for more complex systems.

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We report here that aggregated L L-amyloid (AL L) 1^42 promotes tau aggregation in vitro in a dose-dependent manner. When AL L-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and AL L 1^42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer's disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of AL L 14 2 from these cells was impaired. Taken together, these in vitro results suggest that AL L 1^42 promotes both tau aggregation and hyperphosphorylation. ß