Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.
A molecule isolated from the cerebrospinal fluid of sleep-deprived cats has been chemically characterized and identified as cis-9,10-octadecenoamide. Other fatty acid primary amides in addition to cis-9,10-octadecenoamide were identified as natural constituents of the cerebrospinal fluid of cat, rat, and human, indicating that these compounds compose a distinct family of brain lipids. Synthetic cis-9,10-octadecenoamide induced physiological sleep when injected into rats. Together, these results suggest that fatty acid primary amides may represent a previously unrecognized class of biological signaling molecules.
Full details of the development of a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity are described. The method is based on the loss of fluorescence derived from the displacement of ethidium bromide or thiazole orange from the DNA of interest or, in selected instances, the change in intrinsic fluorescence of a DNA binding agent itself and is applicable for assessing relative or absolute DNA binding affinities. Enlisting a library of hairpin deoxyoligonucleotides containing all five base pair (512 hairpins) or four base pair (136 hairpins) sequences displayed in a 96-well format, a compound's rank order binding to all possible sequences is generated, resulting in a high-resolution definition of its sequence selectivity using this fluorescent intercalator displacement (FID) assay. As such, the technique complements the use of footprinting or affinity cleavage for the establishment of DNA binding selectivity and provides the information at a higher resolution. The merged bar graphs generated by this rank order binding provide a qualitative way to compare, or profile, DNA binding affinity and selectivity. The 96-well format assay (512 hairpins) can be conducted at a minimal cost (presently ca. $100 for hairpin deoxyoligonucleotides/assay with ethiduim bromide or less with thiazole orange), with a rapid readout using a fluorescent plate reader (15 min), and is adaptable to automation (Tecan Genesis Workstation 100 robotic system). Its use in generating a profile of DNA binding selectivity for several agents including distamycin A, netropsin, DAPI, Hoechst 33258, and berenil is described. Techniques for establishing binding constants from quantitative titrations are compared, and recommendations are made for use of a Scatchard or curve fitting analysis of the titration binding curves as a reliable means to quantitate the binding affinity.
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.
Direct-acting cannabinoid receptor agonists are well known to reduce hyperalgesic responses and allodynia after nerve injury, although their psychoactive side effects have damped enthusiasm for their therapeutic development. Alternatively, inhibiting fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), the principal enzymes responsible for the degradation of the respective endogenous cannabinoids, anandamide (AEA) and 2-arachydonylglycerol (2-AG), reduce nociception in a variety of nociceptive assays, with no or minimal behavioral effects. In the present study we tested whether inhibition of these enzymes attenuates mechanical allodynia, and acetone-induced cold allodynia in mice subjected to chronic constriction injury of the sciatic nerve. Acute administration of the irreversible FAAH inhibitor, cyclohexylcarbamic acid 3Ј-carbamoylbiphenyl-3-yl ester (URB597), or the reversible FAAH inhibitor, 1-oxo-1-[5-(2-pyridyl)-2-yl]-7-phenylheptane (OL-135), decreased allodynia in both tests. This attenuation was completely blocked by pretreatment with either CB 1 or CB 2 receptor antagonists, but not by the TRPV1 receptor antagonist, capsazepine, or the opioid receptor antagonist, naltrexone. The novel MAGL inhibitor, 4-nitrophenyl 4-(dibenzo [d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184) also attenuated mechanical and cold allodynia via a CB 1 , but not a CB 2 , receptor mechanism of action. Whereas URB597 did not elicit antiallodynic effects in FAAH(Ϫ/Ϫ) mice, the effects of JZL184 were FAAH-independent. Finally, URB597 increased brain and spinal cord AEA levels, whereas JZL184 increased 2-AG levels in these tissues, but no differences in either endocannabinoid were found between nerve-injured and control mice. These data indicate that inhibition of FAAH and MAGL reduces neuropathic pain through distinct receptor mechanisms of action and present viable targets for the development of analgesic therapeutics.Although cannabis has been used for thousands of years to treat pain and other ailments, its undesirable psychomimetic effects have dampened enthusiasm for further drug development. Instead, recent research has focused on targeting the endogenous cannabinoid system for the development of new analgesics ). The endogenous cannabinoid system consists of two cloned cannabinoid receptors (CB 1 and CB 2 ), various proposed endocannabinoid ligands, including anandamide (AEA; Devane et al., 1992) and 2-arachidonylglycerol (2-AG; Mechoulam et al., 1995), and the enzymes that regulate the biosynthesis and catabolism of the endocannabinoids. In particular, fatty acid amide hydrolase (FAAH;Cravatt et al., 1996) and monoacylglycerol lipase (MAGL;Blankman et al., 2007) are the primary catabolic enzymes of AEA and 2-AG, respectively.
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