Assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LC/MS-based biochemical inhibition of overexpressed DAGLb substrate turnover in vitro
Name: Assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LC/MS-based biochemical inhibition of overexpressed DAGLb substrate turnover in vitro. ..more
BioActive Compounds: 33
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Benjamin Cravatt, The Scripps Research Institute (TSRI)
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 DA025285
Grant Proposal PI: Benjamin Cravatt, The Scripps Research Institute (TSRI)
External Assay ID: DAGLB_INH_LCMS_1%TURNOVER_HTSHITS
Name: Assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LC/MS-based biochemical inhibition of overexpressed DAGLb substrate turnover in vitro.
Endocannabinoids (ECs) represent a unique group of lipids that function as chemical messengers in the nervous system. To date, the two principle ECs identified in mammals are N-arachidonoyl-ethanolamine (anandamide) and 2-arachidonoyl-glycerol (2-AG). They have been implicated in various physiological and pathological functions including appetite, pain, sensation, memory, and addiction (1). Unlike traditional neurotransmitters, which are stored in vesicles, ECs are synthesized and released on demand, and then rapidly degraded to terminate signaling. Thus, the metabolic pathways that govern EC turnover are critical in determining the magnitude and duration of neuronal signaling events (2). Endocannabinoid biosynthesis, in contrast to degradation, is poorly understood. Recently, two serine hydrolases, DAGL-a and DAGL-B, were cloned and found to selectively cleave sn-1 acyl chains from diacylglycerols (DAG) to generate 2-AG in vitro (3). Their function in the nervous system was validated in vivo by the generation of DAGL-a and DAGL-B knock-out mice (4, 5). However, it is still unclear to what extent DAGL-a/B catalytic activity contributes to 2-AG-mediated signaling. The development of potent and selective inhibitors would offer a means to perturb DAGL-a/B activity in a selective, reversible, and temporally-controlled manner. Given the non-selective nature of current DAGL-a/B inhibitors (6), specific chemical probes would serve as invaluable tools to delineate DAGL-a/B function in 2-AG signaling networks of the brain.
1. Di Marzo, V. (2008) Targeting the endocannabinoid system: to enhance or reduce?, Nat Rev Drug Discov 7, 438-455.
2. Ahn, K., McKinney, M. K., and Cravatt, B. F. (2008) Enzymatic pathways that regulate endocannabinoid signaling in the nervous system, Chem Rev 108, 1687-1707.
3. Bisogno, T., Howell, F., Williams, G., Minassi, A., Cascio, M. G., Ligresti, A., Matias, I., Schiano-Moriello, A., Paul, P., Williams, E. J., Gangadharan, U., Hobbs, C., Di Marzo, V., and Doherty, P. (2003) Cloning of the first sn1-DAG lipases points to the spatial and temporal regulation of endocannabinoid signaling in the brain, J Cell Biol 163, 463-468.
4. Gao, Y., Vasilyev, D. V., Goncalves, M. B., Howell, F. V., Hobbs, C., Reisenberg, M., Shen, R., Zhang, M. Y., Strassle, B. W., Lu, P., Mark, L., Piesla, M. J., Deng, K., Kouranova, E. V., Ring, R. H., Whiteside, G. T., Bates, B., Walsh, F. S., Williams, G., Pangalos, M. N., Samad, T. A., and Doherty, P. (2010) Loss of Retrograde Endocannabinoid Signaling and Reduced Adult Neurogenesis in Diacylglycerol Lipase Knock-out Mice, J Neurosci 30, 2017-2024.
5. Tanimura, A., Yamazaki, M., Hashimotodani, Y., Uchigashima, M., Kawata, S., Abe, M., Kita, Y., Hashimoto, K., Shimizu, T., Watanabe, M., Sakimura, K., and Kano, M. (2010) The Endocannabinoid 2-Arachidonoylglycerol Produced by Diacylglycerol Lipase +/- Mediates Retrograde Suppression of Synaptic Transmission, Neuron 65, 320-327.
6. Hoover, H. S., Blankman, J. L., Niessen, S., and Cravatt, B. F. (2008) Selectivity of inhibitors of endocannabinoid biosynthesis evaluated by activity-based protein profiling, Bioorganic & Medicinal Chemistry Letters 18, 5838-5841.
Assay provider, diacylglycerol lipase, diacylglycerol lipase-beta, DAGL, DAGL-beta, DAGLB, hydrolase, serine hydrolase, appetite, pain, sensation, memory, addiction, LC-MS, 1-stearoyl-2-arachidonoylglycerol, SAG, 2-arachidonoylglycerol, 2-AG, substrate, inhibitor, inhibition, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to assess potency of hits from the uHTS assay. This assay relies on the hydrolysis of an endogenous DAG substrate, 1-stearoyl-2-arachidonoylglycerol (SAG), and quantifying the production of 2-arachidonoylglycerol (2-AG) by LC-MS. In this assay, compounds are preincubated with DAGL-beta-transfected HEK293T cell membranes for 30 minutes, followed by incubation with SAG substrate for 30 minutes. The production of 2-AG is quantified by LC-MS. As designed, compounds that act as DAGL-beta inhibitors will slow the rate of enzyme hydrolysis, resulting in a decreased production of 2-AG.
Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGL-b (70 uL of 0.3 mg/mL) in DAGL buffer (5 mM CaCl2, 100 mM NaCl, 50 mM HEPES) was treated with test compound (1.4 uL of a 50x stock in DMSO; 10 uM final concentration) or DMSO (1.4 uL) for 30 minutes at 37 C. The diglyceride substrate, SAG, was added to sample reaction (30 uL, 500 uM final [SAG]) and incubated for 30 minutes at 37 C. The reaction was quenched with 2:1 chloroform/methanol doped with C15:0 monoacylglycerol (MAG) lipid standard (1 nM per 300 uL of quenching solution). The organic (bottom) layer was extracted. 30 uL of the organic phase was injected onto an Agilent 1100 series LC-MSD SL instrument for analysis. LC separation was achieved with a Gemini reversed-phase C18 column (5 um, 4.6 mm x 50 mm). Mobile phase A was composed of 95:5 v/v H2O:MeOH, and mobile phase B was composed of 60:35:5 v/v/v i-PrOH:MeOH:H2O, each containing 0.1% formic acid. The flow rate was 0.5 mL/minute and the gradient consisted of 5 minutes 0% B, a linear increase to 100% B over 15 minutes, followed by an isocratic gradient of 100% B for 12 minutes before equilibrating for 5 minutes at 0% B (37 minutes total). MS analysis, in scanning mode with scanning from m/z = 200-1200, was performed with a positive electrospray ionization (ESI) source. The hydrolysis product 2-AG (379 m/z) was quantified by measuring the area under the peak in comparison with the C15:0 MAG standard (317 m/z) using LC-MS Agilent Chem Station software. The specific activity was calculated by measuring the pmoles of product 2-AG formed per minute per mg of HEK293T-DAGLB proteome used for analysis. The % turnover for each compound was determined by comparing the specific activity of inhibitor-treated proteomes with DMSO-treated proteomes.
Turnover per 0.01 mg per 30 minutes was calculated as follows:
Turnover = AUC_SAMPLE / AUC_STD
AUC_SAMPLE = Product 2-AG AUC for the sample (test compound or control).
AUC_STD = Product 2-AG AUC for the internal standard.
The % Turnover was then calculated as follows:
%_Turnover = ( Turnover_Test_Compound - Median_Turnover_Low_Control ) / ( Median_Turnover_High_Control - Median_TurnoverAUC_Low_Control ) ) * 100
Test_Compound is defined as sample treated with test compound
High_Control is defined as sample treated with DMSO only (no compound).
Low_Control is defined as sample treated with known DAGLb inhibitor THL.
PubChem Activity Outcome and Score:
Compounds with less than or equal to 60% turnover were considered active. Compounds with greater than 60% turnover were considered inactive.
The reported PubChem Activity Score has been normalized to 0% observed turnover.
The PubChem Activity Score range for active compounds is 100-41, and for inactive compounds 40-0.
List of Reagents:
293T Hek cells overexpressing mouse DAGLb (Open Biosystems Accession BC016105; provided by Assay Provider)
SAG (Sigma Aldrich, S6389)
C15:0 MAG (Nu-ChekPrep, M-149)
Gemini C18 reversed phase column (Phenomenex, 00B-4435-E0)
CaCl2 (SigmaAldrich C1016)
NaCl (Fisher Scientific S271)
Hepes (Fisher Scientific AC17257)
Methanol (Fisher Scientific A452)
Chloroform (Fisher Scientific C607)
i-PrOH (Fisher Scientific A451)
Formic acid (Fluka 06440)
This assay was performed by the assay provider with liquid samples of test compounds.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: confirmatory
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay readout content: assay readout method: regular screening
BAO: bioassay specification: assay readout content: content readout type: single readout
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: detection technology: label free technology: mass spectrometry
** Test Concentration.
Data Table (Concise)