The endocannabinoid 2-arachidonoylglycerol (2-AG) is biosynthesized by diacylglycerol lipases DAGLα and DAGLβ. Chemical probes to perturb DAGLs are needed to characterize endocannabinoid function in biological processes. Here, we report a series of in vivo-active 1,2,3-triazole urea inhibitors, along with paired negative-control and activity-based probes, for the functional analysis of DAGLβ in living systems. Optimized inhibitors showed excellent selectivity for DAGLβ over other serine hydrolases, including DAGLα (~60-fold selectivity), and the limited off-targets, such as ABHD6, were also inhibited by the negative-control probe. Using these agents and Daglb−/− mice, we show that DAGLβ inactivation lowers 2-AG, as well as arachidonic acid and eicosanoids, in mouse peritoneal macrophages in a manner that is distinct and complementary to disruption of cytosolic phospholipase-A2 (PLA2G4A). We observed a corresponding reduction in lipopolysaccharide-induced tumor necrosis factor-α release. These findings indicate that DAGLβ is a key metabolic hub within a lipid network that regulates proinflammatory responses in macrophages.

We observed high and persistent spontaneous buildup of the surface potential ͑SP͒ upon vacuum deposition of tris͑8-hydroxyquinolinato͒ aluminum͑III͒ (Alq 3) on an Au substrate under dark conditions. SP determined by the Kelvin probe method reached 28 V at a thickness of 560 nm and the surface of the Alq 3 film was positively charged. We propose a model in which preferential orientation of the dipole moments of Alq 3 molecules is the origin of this buildup of the SP. The intensity of second-harmonic generation was also dramatically increased by the deposition of Alq 3 under dark conditions, which supports the notion of a buildup of dipole layers. This giant surface potential was almost completely removed by irradiation of Alq 3 molecules with visible light, and irradiation during deposition also prevented the buildup of SP.

The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPAL1 and LYPLA2, two enzymes for which selective, in vivo-active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.

Alpha/beta-hydrolase domain containing 6 (ABHD6) is a transmembrane serine hydrolase that hydrolyzes the endogenous cannabinoid 2-arachidonoylglycerol (2-AG) to regulate certain forms of cannabinoid receptor-dependent signaling in the nervous system. The full spectrum of ABHD6 metabolic activities and functions is currently unknown and would benefit from selective, in vivo-active inhibitors. Here, we report the development and characterization of an advanced series of irreversible (2-substituted)-piperidyl-1,2,3-triazole urea inhibitors of ABHD6, including compounds KT182 and KT203, which show exceptional potency and selectivity in cells (< 5 nM) and, at equivalent doses in mice (1 mg kg-1), served as systemic and peripherally-restricted ABHD6 inhibitors, respectively. We also describe an orally-bioavailable ABHD6 inhibitor KT185 that displays excellent selectivity against other brain and liver serine hydrolases in vivo. We thus describe several chemical probes for biological studies of ABHD6, including brain-penetrant and peripherally-restricted inhibitors that should prove of value for interrogating ABHD6 function in animal models.

Glutathione S-transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Glutathione S-tranferase omega 1 (GSTO1) is highly expressed in human cancer cells, where it has been suggested to play a role in detoxification of chemotherapeutic agents. Selective inhibitors of GSTO1 are, however, required to test the role that this enzyme plays in cancer and other (patho)physiological processes. With this goal in mind, we performed a fluorescence polarization activity-based protein profiling (fluopol-ABPP) high-throughput screen (HTS) with GSTO1 and the Molecular Libraries Small Molecule Repository (MLSMR) 300K+ compound library. This screen identified a class of selective and irreversible α-chloroacetamide inhibitors of GSTO1, which were optimized to generate an agent KT53 that inactivates GSTO1 with excellent in vitro (IC50 = 21 nM) and in situ (IC50 = 35 nM) potency. Cancer cells treated with KT53 show heightened sensitivity to the cytotoxic effects of cisplatin, supporting a role for GSTO1 in the detoxification of chemo-therapeutic agents

We have previously shown that 1,2,3-triazole ureas (1,2,3-TUs) act as versatile class of irreversible serine hydrolase inhibitors that can be tuned to create selective probes for diverse members of this large enzyme class, including diacylglycerol lipase-β (DAGLβ), a principal biosynthetic enzyme for the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we provide a detailed account of the discovery, synthesis, and structure-activity relationship (SAR) of (2-substituted)-piperidyl-1,2,3-TUs that selectively inactivate DAGLβ in living systems. Key to success was the use of activity-based protein profiling (ABPP) with broad-spectrum and tailored activity-based probes to guide our medicinal chemistry efforts. We also describe an expanded repertoire of DAGL-tailored activity-based probes that includes biotinylated and alkyne agents for enzyme enrichment coupled with mass spectrometry-based proteomics and assessment of proteome-wide selectivity. Our findings highlight the broad utility of 1,2,3-TUs for serine hydrolase inhibitor development and their application to create selective probes of endocannabinoid biosynthetic pathways.

Donor-acceptor chromophores were almost quantitatively introduced into the side chains of a polystyrene derivative by sequential "click chemistry"-type addition reactions as an efficient postfunctionalization method. The first click reaction is the conventional Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and the second one is the atom-economic addition of strong acceptor molecules, such as tetracyanoethylene (TCNE) and 7,7,8,8-tetracyanoquinodimethane (TCNQ), to the aniline-substituted electron-rich alkynes. Steric hindrance was found to be an important factor in determining the reactivity of alkyne-acceptor addition reactions. All obtained polymers showed good solubility in common organic solvents, and they were fully characterized by GPC, 1 H NMR and IR spectroscopy, and elemental analysis. After the acceptor addition, the polymers showed an intense charge-transfer (CT) band centered at ca. 480 nm for the TCNE adducts and ca. 710 nm for the TCNQ adducts. Electrochemical measurements of these polymers also revealed well-defined oxidation and reduction potentials, offering consistency between the electrochemical and optical band gaps. The second harmonic generation (SHG) coefficients (d 33 and d 15 ) of the polymer thin films were evaluated by SHG measurements before and after corona poling at 150 °C, a temperature that was determined on the basis of thermal analyses. The results show that the TCNE adducted polymers possess better SHG properties than the corresponding TCNQ adducted polymers, probably reflecting the superior chromophore mobility within the polymers.

Multiwalled carbon nanotube (MWNT)/poly(vinyl butyral) (PVB) composite nanofibers were prepared by electrospinning, successive twisting and heat treatment. The MWNTs were highly oriented in an electrified thin jet during electrospinning. The heat treatment of the twisted electrospun nanofiber yarns produced the characteristics of the CNT in the composite nanofiber yarns and enhanced their electrical properties, mechanical properties, and thermal properties. The electrical conductivity of the heated yarn was significantly enhanced and showed the maximum value of 154 S cm(-1) for the yarn heated at 400 °C. It is an order of magnitude higher than other electrospun CNT composite materials. These results demonstrated that the novel top-down process based on electrospinning, twisting, and heat treatment provide a promising option for simple and large-scale manufacture of CNT assemblies.