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BioAssay: AID 624468

Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assay

Name: Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assay. ..more
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 Tested Compounds
 Tested Compounds
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Active(2)
 
 
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 Tested Substances
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Active(2)
 
 
AID: 624468
Data Source: The Scripps Research Institute Molecular Screening Center (DAGLB_INH_LCMS_2XIC50)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-08-08
Hold-until Date: 2012-10-12
Modify Date: 2012-10-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
504411Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of human diacylglycerol lipase, beta (DAGLB)Screening depositor-specified cross reference: Primary screen (DAGLB inhibitors in singlicate)
504420Summary of the probe development efforts to identify inhibitors of human diacylglycerol lipase, beta (DAGLB)Summary3 depositor-specified cross reference: Summary (DAGLB inhibitors)
504445Fluorescence-based biochemical high throughput confirmation assay for inhibitors of human diacylglycerol lipase, beta (DAGLB)Screening depositor-specified cross reference: Confirmation screen (DAGLB inhibitors in triplicate)
602299Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of overexpressed DAGLb in vitro set 2Other depositor-specified cross reference: Late stage assay (overexpressed DAGLb inhibitors in vitro)
602300Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of overexpressed DAGLb in vitro set 3Other depositor-specified cross reference: Late stage assay (overexpressed DAGLb inhibitors in vitro)
602301Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of recombinant anti-target ABHD11 in vitro set 1Other depositor-specified cross reference: Late stage assay (ABHD11 inhibitors)
602302Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of overexpressed DAGLb in vitro set 1Other depositor-specified cross reference: Late stage assay (overexpressed DAGLb inhibitors in vitro)
602303Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of overexpressed DAGLb in vitro; triazole urea libraryOther depositor-specified cross reference: Late stage assay (overexpressed DAGLb inhibitors in vitro; triazole urea library)
602311Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of serine hydrolases in mouse brain membrane in vitro set 1Other depositor-specified cross reference: Late stage assay (serine hydrolases in mouse brain membrane inhibitors in vitro)
602312Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of recombinant anti-target ABHD11 in vitro set 2Other depositor-specified cross reference: Late stage assay (ABHD11 inhibitors in vitro)
602319Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of antitarget ABHD6 in vitroOther depositor-specified cross reference: Late stage assay (ABHD6 inhibitors in vitro)
602320Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based dose-response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of DAGLb in vitroConfirmatory depositor-specified cross reference: Late stage assay (DAGLb inhibitors in vitro in triplicate)
602321Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of serine hydrolases in mouse brain membrane in vitro set 3Other depositor-specified cross reference: Late stage assay (serine hydrolases in mouse brain membrane inhibitors in vitro)
602322Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based dose-response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of antitarget ABHD6 in vitroConfirmatory depositor-specified cross reference: Late stage assay (ABHD6 inhibitors in vitro in triplicate)
602323Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of serine hydrolases in mouse brain membrane in vitro set 2Other depositor-specified cross reference: Late stage assay (serine hydrolases in mouse brain membrane inhibitors in vitro)
602335Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based dose-response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of anti-target ABHD6 in situConfirmatory depositor-specified cross reference: Late stage assay (ABHD6 inhibitors in situ in triplicate)
602337Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Late stage dose response assay (cytotoxicity in five replicates)
602339Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysisOther depositor-specified cross reference: Late stage assay (DAGLb SILAC selectivity analysis)
602341Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based cell-based Activity-Based Protein Profiling (ABPP) SILAC selectivity analysis for ABHD6Other depositor-specified cross reference: Late stage assay (ABHD6 SILAC selectivity analysis)
602343Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of mouse brain ABHD6 in vivo, set 2Other depositor-specified cross reference: Late stage assay (mouse brain ABHD6 inhibitors in vivo)
602345Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of mouse macrophage DAGLb in vivoOther depositor-specified cross reference: Late stage assay (mouse macrophage DAGLb inhibitors in vivo in duplicate)
602347Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of mouse brain ABHD6 in vivoOther depositor-specified cross reference: Late stage assay (mouse brain ABHD6 inhibitors in vivo in duplicate)
602349Late stage assay provider results from the probe development effort to identify inhibitors of DAGLb: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) assay to distinguish systemic and peripheral inhibitorsOther depositor-specified cross reference: Late stage assay (distinguish systemic and peripheral inhibitors in duplicate)
602351Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based animal-based Activity-Based Protein Profiling (ABPP) MudPIT selectivity analysis for ABHD6Other depositor-specified cross reference: Late stage assay (ABHD6 MudPIT selectivity analysis in triplicate)
602353Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based animal-based Activity-Based Protein Profiling (ABPP) MudPIT selectivity analysisOther depositor-specified cross reference: Late stage assay (DAGLb MudPIT selectivity analysis in triplicate)
602354Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical dose-response gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of DAGLb in situConfirmatory depositor-specified cross reference: Late stage dose-response (DAGLb inhibitors in situ in triplicate)
602355Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of serine hydrolases in vitroOther depositor-specified cross reference: Late stage assay (serine hydrolases inhibitors in vitro)
602403Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of recombinant antitarget DAGLa in vitroOther depositor-specified cross reference: Late stage assay (recombinant DAGLa inhibitors in vitro)
602415Assay 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 vitroOther depositor-specified cross reference: Late stage assay (DAGLb substrate turnover inhibitors in vitro)
624039Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based dose-response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of antitarget ABHD6 in vitro, set 2Confirmatory depositor-specified cross reference: Late stage assay (ABHD6 inhibitors in vitro)
624041Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of mouse liver ABHD6 in vivoOther depositor-specified cross reference: Late stage assay (mouse liver ABHD6 inhibitors in vivo)
624077Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of mouse liver ABHD6 in vivo upon oral compound administrationOther depositor-specified cross reference: Late stage assay (mouse liver ABHD6 inhibitors in vivo upon oral compound administration)
624472Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of DAGLb by enantiomers of KT116Other same project related to Summary assay
Description:
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_2XIC50

Name: Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assay.

Description:

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 -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 -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.

References:

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.

Keywords:

late stage, late stage AID, assay provider, powders, dose response, 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, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:
The purpose of this assay is to determine the IC50 of hit compounds in a biochemical assay based on hydrolysis of an endogenous DAG substrate, 1-stearoyl-2-arachidonoylglycerol (SAG), and quantification of 2-arachidonoylglycerol (2-AG) production 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. Text compounds were assayed in duplicate in a 6-point dilution series starting at a nominal concentration of 10 uM.
Protocol Summary:
Membrane proteome of transiently transfected 293T Hek cells overexpressing mouse DAGLb (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 concentration) 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 nmol 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/min 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 from 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). 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 percent inhibition 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
Where:
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 - MedianTurnover_Low_Control ) / ( MedianTurnover_High_Control - MedianTurnoverAUC_Low_Control ) ) * 100
Where:
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.
For each test compound, percent turnover was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.
PubChem Activity Outcome and Score:
Compounds with an IC50 value of less than 0.500 uM were considered active. Compounds with an IC50 value greater than or equal to 0.500 uM were considered inactive.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
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)
Millipore-filtered water
Formic acid (Fluka 06440)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
2Turnover at 10 uM [1] (10μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 10 uM compound treatment as assessed by LC-MS.Float%
3Turnover at 2 uM [1] (2μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 2 uM compound treatment as assessed by LC-MS.Float%
4Turnover at 0.4 uM [1] (0.4μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.4 uM compound treatment as assessed by LC-MS.Float%
5Turnover at 0.08 uM [1] (0.08μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.08 uM compound treatment as assessed by LC-MS.Float%
6Turnover at 0.016 uM [1] (0.016μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.016 uM compound treatment as assessed by LC-MS.Float%
7Turnover at 0.0032 uM [1] (0.032μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.0032 uM compound treatment as assessed by LC-MS.Float%
8Turnover at 10 uM [2] (10μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 10 uM compound treatment as assessed by LC-MS.Float%
9Turnover at 2 uM [2] (2μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 2 uM compound treatment as assessed by LC-MS.Float%
10Turnover at 0.4 uM [2] (0.4μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.4 uM compound treatment as assessed by LC-MS.Float%
11Turnover at 0.08 uM [2] (0.08μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.08 uM compound treatment as assessed by LC-MS.Float%
12Turnover at 0.016 uM [2] (0.016μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.016 uM compound treatment as assessed by LC-MS.Float%
13Turnover at 0.0032 uM [2] (0.032μM**)% turnover of SAG substrate (as monitored by 2-AG product formation) of overexpressed mouse DAGLb upon 0.0032 uM compound treatment as assessed by LC-MS.Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R01 DA025285

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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