The endocannabinoids (eCBs) anandamide and 2-arachidonoyl glycerol (2-AG) are inactivated by a two-step mechanism. First, they are carried into cells, and then anandamide is hydrolyzed by fatty acid amide hydrolase (FAAH) and 2-AG by monoacylglycerol lipase (MGL). Here we provide evidence for a previously undescribed MGL activity expressed by microglial cells. We found that the mouse microglial cell line BV-2 does not express MGL mRNA and yet efficiently hydrolyzes 2-AG. URB597 (3Ј-carbamoyl-biphenyl-3-ylcyclohexylcarbamate) reduces this hydrolysis by 50%, suggesting the involvement of FAAH. The remaining activity is blocked by classic MGL inhibitors [[1,1-biphenyl]-3-yl-carbamic acid, cyclohexyl ester (URB602) and MAFP (methylarachidonyl fluorophosphate)] and is unaffected by inhibitors of COXs (cyclooxygenases), LOXs (lipooxygenases), and DGLs (diacylglycerol lipases), indicating the involvement of a novel MGL activity. Accordingly, URB602 leads to selective accumulation of 2-AG in intact BV-2 cells. Although MGL expressed in neurons is equally distributed between the cytosolic, mitochondrial, and nuclear fractions, the novel MGL activity expressed by BV-2 cells is enriched in mitochondrial and nuclear fractions. A screen for novel inhibitors of eCB hydrolysis identified several compounds that differentially block MGL, FAAH, and the novel MGL activity. Finally, we provide evidence for expression of the novel MGL by mouse primary microglia in culture. Our results suggest the presence of a novel, pharmacologically distinct, MGL activity that controls 2-AG levels in microglia.

Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

X-ray crystal structures at 2.9 Å resolution are reported for complexes of catechol diethers 1 and 2 with HIV-1 reverse transcriptase. The results help elucidate the structural origins of the extreme antiviral activity of the compounds. The possibility of halogen bonding between the inhibitors and Pro95 is addressed. Structural analysis reveals key interactions with conserved residues P95 and W229 of importance for design of inhibitors with high potency and favorable resistance profiles.

Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders.

Endocannabinoids have recently emerged as instructive cues in the developing central nervous system, and, based on the expression of their receptors, we identified oligodendrocytes as potential targets of these molecules. Here, we show that the enzymes responsible for the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG), diacylglycerol lipase alpha (DAGLα) and beta (DAGLβ), and degradation, monoacylglycerol lipase (MAGL), can be found in oligodendrocytes at different developmental stages. Moreover, cultured oligodendrocyte progenitor cells (OPCs) express DAGLα and β abundantly, resulting in the stronger production of 2-AG than in differentiated oligodendrocytes. The opposite is observed with MAGL. CB1 and CB2 receptor antagonists (SR141716 and AM630) impaired OPC differentiation into mature oligodendrocytes and likewise, inhibiting DAGL activity with RHC-80267 or tetrahydrolipstatin also blocked oligodendrocyte maturation, an effect reversed by the addition of exogenous 2-AG. Likewise, 2-AG synthesis disruption using specific siRNAs against DAGLα and DAGLβ significantly reduced myelin protein expression in vitro, whereas a pharmacological gain-of-function approach by using cannabinoid agonists or MAGL inhibition had the opposite effects. ERK/MAPK pathway is implicated in oligodendrocyte differentiation because PD98059, an inhibitor of MEK1, abrogated oligodendrocyte maturation. The cannabinoid receptor antagonists and RHC-80267 all diminished basal ERK1/2 phosphorylation, effects that were partially reversed by the addition of 2-AG. Overall, our data suggest a novel role of endocannabinoids in oligodendrocyte differentiation such that constitutive release of 2-AG activates cannabinoid receptors in an autocrine/paracrine way in OPCs, stimulating the ERK/MAPK signaling pathway.