Matthew P. Patricelli

Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase

The medicinal properties of marijuana have been recognized for centuries, but clinical and societal acceptance of this drug of abuse as a potential therapeutic agent remains fiercely debated. An attractive alternative to marijuana-based therapeutics would be to target the molecular pathways that mediate the effects of this drug. To date, these neural signaling pathways have been shown to comprise a cannabinoid receptor (CB1) that binds the active constituent of marijuana, tetrahydrocannabinol (THC), and a postulated endogenous CB1 ligand anandamide. Although anandamide binds and activates the CB1 receptor in vitro, this compound induces only weak and transient cannabinoid behavioral effects in vivo, possibly a result of its rapid catabolism. Here we show that mice lacking the enzyme fatty acid amide hydrolase (FAAH−/−) are severely impaired in their ability to degrade anandamide and when treated with this compound, exhibit an array of intense CB1-dependent behavioral responses, including hypomotility, analgesia, catalepsy, and hypothermia. FAAH−/−-mice possess 15-fold augmented endogenous brain levels of anandamide and display reduced pain sensation that is reversed by the CB1 antagonist SR141716A. Collectively, these results indicate that FAAH is a key regulator of anandamide signaling in vivo, setting an endogenous cannabinoid tone that modulates pain perception. FAAH may therefore represent an attractive pharmaceutical target for the treatment of pain and neuropsychiatric disorders.

Characterization of a selective inhibitor of the Parkinson's disease kinase LRRK2

Mutations in leucine-rich repeat kinase 2 (LRRK2) are strongly associated with late-onset autosomal dominant Parkinson’s disease. We employed a novel, parallel, compound-centric approach to identify a potent and selective LRRK2 inhibitor LRRK2-IN-1, and demonstrated that inhibition of LRRK2 induces dephosphorylation of Ser910/Ser935 and accumulation of LRRK2 within aggregate structures. LRRK2-IN-1 will serve as a versatile tool to pharmacologically interrogate LRRK2 biology and study its role in Parkinson’s disease.

Direct visualization of serine hydrolase activities in complex proteomes using fluorescent active site‐directed probes

The field of biochemistry is currently faced with the enormous challenge of assigning functional significance to more than thirty thousand predicted protein products encoded by the human genome. In order to accomplish this daunting task, methods will be required that facilitate the global analysis of proteins in complex biological systems. Recently, methods have been described for simultaneously monitoring the activity of multiple enzymes in crude proteomes based on their reactivity with tagged chemical probes. These activity based probes (ABPs) have used either radiochemical or biotin/avidin‐based detection methods to allow consolidated visualization of numerous enzyme activities. Here we report the synthesis and evaluation of fluorescent activity based probes for the serine hydrolase super‐family of enzymes. The fluorescent methods detailed herein provide superior throughput, sensitivity, and quantitative accuracy when compared to previously described ABPs, and provide a straightforward platform for high‐throughput proteome analysis.

Reversible inhibitor of p97, DBeQ, impairs both ubiquitin-dependent and autophagic protein clearance pathways

A specific small-molecule inhibitor of p97 would provide an important tool to investigate diverse functions of this essential ATPase associated with diverse cellular activities (AAA) ATPase and to evaluate its potential to be a therapeutic target in human disease. We carried out a high-throughput screen to identify inhibitors of p97 ATPase activity. Dual-reporter cell lines that simultaneously express p97-dependent and p97-independent proteasome substrates were used to stratify inhibitors that emerged from the screen. N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) was identified as a selective, potent, reversible, and ATP-competitive p97 inhibitor. DBeQ blocks multiple processes that have been shown by RNAi to depend on p97, including degradation of ubiquitin fusion degradation and endoplasmic reticulum-associated degradation pathway reporters, as well as autophagosome maturation. DBeQ also potently inhibits cancer cell growth and is more rapid than a proteasome inhibitor at mobilizing the executioner caspases-3 and -7. Our results provide a rationale for targeting p97 in cancer therapy.

Trifluoromethyl ketone inhibitors of fatty acid amide hydrolase: a probe of structural and conformational features contributing to inhibition.

The examination of a series of trifluoromethyl ketone inhibitors of Fatty Acid Amide Hydrolase (FAAH, oleamide hydrolase, anandamide amidohydrolase) is detailed in efforts that define structural and conformational properties that contribute to enzyme inhibition and substrate binding. The results imply an extended bound conformation, highlight a role for the presence, position, and stereochemistry of a delta cis double bond, and suggest little apparent role for C11-C18/C22 of the fatty acid amide substrates.

Fatty acid amide hydrolase substrate specificity.

Fatty acid amide hydrolase (FAAH), also referred to as oleamide hydrolase and anandamide amidohydrolase, is a serine hydrolase responsible for the degradation of endogenous oleamide and anandamide, fatty acid amides that function as chemical messengers. FAAH hydrolyzes a range of fatty acid amides, and the present study examines the relative rates of hydrolysis of a variety of natural and unnatural fatty acid primary amide substrates using pure recombinant rat FAAH.

Functional Interplay between Caspase Cleavage and Phosphorylation Sculpts the Apoptotic Proteome

Caspase proteases are principal mediators of apoptosis, where they cleave hundreds of proteins. Phosphorylation also plays an important role in apoptosis, although the extent to which proteolytic and phosphorylation pathways crosstalk during programmed cell death remains poorly understood. Using a quantitative proteomic platform that integrates phosphorylation sites into the topographical maps of proteins, we identify a cohort of over 500 apoptosis-specific phosphorylation events and show that they are enriched on cleaved proteins and clustered around sites of caspase proteolysis. We find that caspase cleavage can expose new sites for phosphorylation, and, conversely, that phosphorylation at the +3 position of cleavage sites can directly promote substrate proteolysis by caspase-8. This study provides a global portrait of the apoptotic phosphoproteome, revealing heretofore unrecognized forms of functional crosstalk between phosphorylation and caspase proteolytic pathways that lead to enhanced rates of protein cleavage and the unveiling of new sites for phosphorylation.

Kinome-wide Selectivity Profiling of ATP-competitive Mammalian Target of Rapamycin (mTOR) Inhibitors and Characterization of Their Binding Kinetics

Background: Several new ATP-competitive mTOR inhibitors have been described, but their kinome-wide selectivity profiles have not been disclosed. Results: Four different profiling technologies revealed a different spectrum of targets for four recently described mTOR inhibitors. Conclusion: Diverse heterocyclic mTOR inhibitors have unique pharmacology. Significance: Profiling data guide choices of mTOR inhibitors for particular applications and provide new potential targets for medicinal chemistry efforts. An intensive recent effort to develop ATP-competitive mTOR inhibitors has resulted in several potent and selective molecules such as Torin1, PP242, KU63794, and WYE354. These inhibitors are being widely used as pharmacological probes of mTOR-dependent biology. To determine the potency and specificity of these agents, we have undertaken a systematic kinome-wide effort to profile their selectivity and potency using chemical proteomics and assays for enzymatic activity, protein binding, and disruption of cellular signaling. Enzymatic and cellular assays revealed that all four compounds are potent inhibitors of mTORC1 and mTORC2, with Torin1 exhibiting ∼20-fold greater potency for inhibition of Thr-389 phosphorylation on S6 kinases (EC50 = 2 nm) relative to other inhibitors. In vitro biochemical profiling at 10 μm revealed binding of PP242 to numerous kinases, although WYE354 and KU63794 bound only to p38 kinases and PI3K isoforms and Torin1 to ataxia telangiectasia mutated, ATM and Rad3-related protein, and DNA-PK. Analysis of these protein targets in cellular assays did not reveal any off-target activities for Torin1, WYE354, and KU63794 at concentrations below 1 μm but did show that PP242 efficiently inhibited the RET receptor (EC50, 42 nm) and JAK1/2/3 kinases (EC50, 780 nm). In addition, Torin1 displayed unusually slow kinetics for inhibition of the mTORC1/2 complex, a property likely to contribute to the pharmacology of this inhibitor. Our results demonstrated that, with the exception of PP242, available ATP-competitive compounds are highly selective mTOR inhibitors when applied to cells at concentrations below 1 μm and that the compounds may represent a starting point for medicinal chemistry efforts aimed at developing inhibitors of other PI3K kinase-related kinases.

Proteins regulating the biosynthesis and inactivation of neuromodulatory fatty acid amides

Fatty acid amides (FAAs) represent a growing family of biologically active lipids implicated in a diverse range of cellular and physiological processes. At present, two general types of fatty acid amides, the N-acylethanolamines (NAEs) and the fatty acid primary amides (FAPAs), have been identified as potential physiological neuromodulators/neurotransmitters in mammals. Representative members of these two subfamilies include the endocannabinoid NAE anandamide and the sleep-inducing FAPA oleamide. In this Chapter, molecular mechanisms proposed for the biosynthesis and inactivation of FAAs are critically evaluated, with an emphasis placed on the biochemical and cell biological properties of proteins thought to mediate these processes.

AN ENDOGENOUS SLEEP-INDUCING COMPOUND IS A NOVEL COMPETITIVE INHIBITOR OF FATTY ACID AMIDE HYDROLASE

2-Octyl gamma-bromoacetoacetate (O gamma Br), an endogenous compound originally isolated from human cerebrospinal fluid (CSF), has previously been demonstrated to increase REM sleep duration in cats. Based on the chemical structure of O gamma Br and its reported sleep-inducing effects, we synthesized O gamma Br along with chemically related analogs and tested these compounds as inhibitors of fatty acid amide hydrolase (FAAH), a brain enzyme that degrades neuromodulatory fatty acid amides. O gamma Br was found to competitively inhibit FAAH activity with IC50 and Ki values of 2.6 microM and 0.8 microM, respectively [for the (R)-enantiomer of O gamma Br (1)]. A set of synthetic analogs of O gamma Br was examined to define the structural features required for FAAH inhibition and inhibitor potencies were assessed at pH 9.0 (near the pH optimum of FAAH) and pH 7.0. Interestingly, at pH 7.0 the gamma-halo beta-keto ester inhibitors proved to be significantly more potent than the trifluoromethyl ketone of oleic acid, one of the most potent FAAH inhibitors described to date. This study supports the possibility that O gamma Br may be a physiological regulator of FAAH activity and fatty acid amide levels in vivo. Additionally, the characterization of gamma-halo beta-keto esters as powerful FAAH inhibitors near physiological pH may aid in future studies of the enzymology and biological properties of FAAH.