ol-135 and anandamide

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme within the amidase-signature family. It catalyzes the hydrolysis of several endogenous biologically active lipids, including anandamide (arachidonoyl ethanolamide), oleoyl ethanolamide, and palmitoyl ethanolamide. These endogenous FAAH substrates have been shown to be involved in a variety of physiological and pathological processes, including synaptic regulation, regulation of sleep and feeding, locomotor activity, pain and inflammation. Here we describe the biochemical and biological properties of a potent and selective FAAH inhibitor, 4-(3-phenyl-[1,2,4]thiadiazol-5-yl)-piperazine-1-carboxylic acid phenylamide (JNJ-1661010). The time-dependence of apparent IC(50) values at rat and human recombinant FAAH, dialysis and mass spectrometry data indicate that the acyl piperazinyl fragment of JNJ-1661010 forms a covalent bond with the enzyme. This bond is slowly hydrolyzed, with release of the piperazinyl fragment and recovery of enzyme activity. The lack of inhibition observed in a rat liver esterase assay suggests that JNJ-1661010 is not a general esterase inhibitor. JNJ-1661010 is >100-fold preferentially selective for FAAH-1 when compared to FAAH-2. JNJ-1661010 dose-dependently increases arachidonoyl ethanolamide, oleoyl ethanolamide, and palmitoyl ethanolamide in the rat brain. The compound attenuates tactile allodynia in the rat mild thermal injury model of acute tissue damage and in the rat spinal nerve ligation (Chung) model of neuropathic pain. JNJ-1661010 also diminishes thermal hyperalgesia in the inflammatory rat carrageenan paw model. These data suggest that FAAH inhibitors with modes of action similar to JNJ-1661010 may be useful clinically as broad-spectrum analgesics.

Topics: Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Brain; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Hot Temperature; Humans; Hydrolysis; Isoenzymes; Kinetics; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neuralgia; Oleic Acids; Pain; Pain Measurement; Pain Threshold; Palmitic Acids; Piperazines; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reaction Time; Recombinant Proteins; Thiadiazoles

A study of the structure-activity relationships (SAR) of 2f (OL-135), a potent inhibitor of fatty acid amide hydrolase (FAAH), is detailed, targeting the 5-position of the oxazole. Examination of a series of substituted benzene derivatives (12-14) revealed that the optimal position for substitution was the meta-position with selected members approaching or exceeding the potency of 2f. Concurrent with these studies, the effect of substitution on the pyridine ring of 2f was also examined. A series of small, nonaromatic C5-substituents was also explored and revealed that the K(i) follows a well-defined correlation with the Hammett sigma(p) constant (rho = 3.01, R2 = 0.91) in which electron-withdrawing substituents enhance potency, leading to inhibitors with K(i)s as low as 400 pM (20n). Proteomic-wide screening of the inhibitors revealed that most are exquisitely selective for FAAH over all other mammalian proteases, reversing the 100-fold preference of 20a (C5 substituent = H) for the enzyme TGH.

Topics: Amidohydrolases; Animals; Arachidonic Acids; Benzene Derivatives; Chlorocebus aethiops; COS Cells; Endocannabinoids; Humans; Oleic Acids; Oxazoles; Polyunsaturated Alkamides; Proteomics; Rats; Recombinant Proteins; Structure-Activity Relationship

Fatty acid amides constitute a large and diverse class of lipid transmitters that includes the endogenous cannabinoid anandamide and the sleep-inducing substance oleamide. The magnitude and duration of fatty acid amide signaling are controlled by enzymatic hydrolysis in vivo. Fatty acid amide hydrolase (FAAH) activity in mammals has been primarily attributed to a single integral membrane enzyme of the amidase signature (AS) family. Here, we report the functional proteomic discovery of a second membrane-associated AS enzyme in humans that displays FAAH activity. The gene that encodes this second FAAH enzyme was found in multiple primate genomes, marsupials, and more distantly related vertebrates, but, remarkably, not in a number of lower placental mammals, including mouse and rat. The two human FAAH enzymes, which share 20% sequence identity and are referred to hereafter as FAAH-1 and FAAH-2, hydrolyzed primary fatty acid amide substrates (e.g. oleamide) at equivalent rates, whereas FAAH-1 exhibited much greater activity with N-acyl ethanolamines (e.g. anandamide) and N-acyl taurines. Both enzymes were sensitive to the principal classes of FAAH inhibitors synthesized to date, including O-aryl carbamates and alpha-keto heterocycles. These data coupled with the overlapping, but distinct tissue distributions of FAAH-1 and FAAH-2 suggest that these proteins may collaborate to control fatty acid amide catabolism in primates. The apparent loss of the FAAH-2 gene in some lower mammals should be taken into consideration when extrapolating genetic or pharmacological findings on the fatty acid amide signaling system across species.

Topics: Amidohydrolases; Amino Acid Sequence; Animals; Arachidonic Acids; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Endocannabinoids; Fatty Acids; Humans; Hydrolysis; Mice; Molecular Sequence Data; Placenta; Polyunsaturated Alkamides; Rats; Sequence Homology, Amino Acid; Signal Transduction; Tissue Distribution