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BioAssay: AID 624472

Late 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 KT116

Name: Late 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 KT116. ..more
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 Tested Compounds
 Tested Compounds
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Active(1)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(2)
 
 
Inactive(1)
 
 
AID: 624472
Data Source: The Scripps Research Institute Molecular Screening Center (DAGLB_INH_FLUO_1X%INH_ENANTIOMERS_KT116)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-08-08
Hold-until Date: 2012-10-15
Modify Date: 2012-10-15

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
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)
624468Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assayConfirmatory 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_FLUO_1X%INH_ENANTIOMERS_KT116

Name: Late 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 KT116.

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, enantiomers, diacylglycerol lipase, diacylglycerol lipase-beta, DAGL, DAGL-beta, DAGLB, hydrolase, serine hydrolase, appetite, pain, sensation, memory, addiction, abhydrolase domain containing protein 6, ABHD6, activity-based protein profiling, ABPP, gel-based, activity-based probe, HT-01, inhibitor, inhibition, Neuro-2A, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Assays
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1DAGLb21sn1-specific diacylglycerol lipase beta [Mus musculus] [gi:31559956]
Taxonomy id: 10090
Gene id: 231871
2ABHD621monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]
Taxonomy id: 10090
Gene id: 66082

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to determine whether enantiomers of a test compound can inhibit DAGLb and ABHD6 in a gel-based competitive activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome containing DAGLb and ABHD6 is incubated with test compound followed by reaction with a fluorescently-labeled activity-based probe, HT-01, which selectively labels several serine hydrolases including DAGLb and ABHD6. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density (IOD) of the bands. As designed, test compounds that act as inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.

Protocol Summary:

Membrane proteome of Neuro-2A murine neuroblastoma cells (25 uL of 1 mg/mL) in Dulbecco's PBS (DPBS) was treated with test compound (0.5 uL of a 50x stock in DMSO; 0.001 uM, 0.010 uM, 0.100 uM, 1.000 uM, 10.000 uM [1 nM, 10 nM, 100 nM, 1000 nM, or 10000 nM] final concentration) or DMSO (0.5 uL) for 30 minutes at 37 C. The activity-based probe HT-01 (0.5 uL of a 50x stock in DMSO; 1 uM final concentration) was added, and the reaction was incubated for 30 minutes at 37 C, quenched with an equal volume of 2x SDS-PAGE loading buffer (reducing), separated by SDS-PAGE, and visualized by in-gel fluorescent scanning. The percentage of DAGLb and ABHD6 activity remaining was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control.

The percent inhibition for each compound was calculated as follows:

%_Inhibition = (1-(IOD_Test_Compound-MedianIOD_Low_Control)/(MedianIOD_High_Control-MedianIOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as complex proteome treated with test compound.
High_Control is defined as complex proteome treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

PubChem Activity Outcome and Score:

The following applies to each panel in this assay:

Compounds with greater than or equal to 50% inhibition at 0.100 uM test compound concentration were considered active. Compounds with less than 50% inhibition at 0.100 uM test compound concentration were considered inactive.

The reported PubChem Activity Score has been normalized to 100% of the observed value at 0.100 uM.

DAGLb Score: The PubChem Activity Score range for active compounds is 100-64, and for inactive compounds 0-0.

ABHD6 Score: The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 10-10.

Overall Outcome and Score:

Compounds active against both DAGLb and ABHD6 were considered active. Compounds inactive against DAGLb and/or ABHD6 were considered inactive.

The PubChem Activity Score is assigned a value of 100 for active compounds, and 0 for inactive compounds.

The PubChem Activity Score range for active compounds is 100-100, and for inactive compounds 0-0.

List of Reagents:

Neuro-2A proteome (provided by Assay Provider)
HT-01 (provided by Assay Provider)
DPBS (Cellgro 20-031-CV)
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
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Outcome [DAGLb]One of Active, Inactive, or Not Tested for DAGLb assay1sn1-specific diacylglycerol lipase beta [Mus musculus]Outcome
2Score [DAGLb]The BioAssay activity ranking score1Integer
3Inhibition at 10.000 uM [DAGLb] (10μM**)Inhibition of endogenous mouse DAGLb upon 10000 nM compound treatment as assessed by gel-based competitive ABPP.1Integer%
4Inhibition at 1.000 uM [DAGLb] (1μM**)Inhibition of endogenous mouse DAGLb upon 1000 nM compound treatment as assessed by gel-based competitive ABPP.1Integer%
5Inhibition at 0.100 uM [DAGLb] (0.1μM**)Inhibition of endogenous mouse DAGLb upon 100 nM compound treatment as assessed by gel-based competitive ABPP.1Integer%
6Inhibition at 0.010 uM [DAGLb] (0.01μM**)Inhibition of endogenous mouse DAGLb upon 10 nM compound treatment as assessed by gel-based competitive ABPP.1Integer%
7Inhibition at 0.001 uM [DAGLb] (0.001μM**)Inhibition of endogenous mouse DAGLb upon 1 nM compound treatment as assessed by gel-based competitive ABPP.1Integer%
8Outcome [ABHD6]One of Active, Inactive, or Not Tested for ABHD6 assay2monoacylglycerol lipase ABHD6 [Mus musculus]Outcome
9Score [ABHD6]The BioAssay activity ranking score2Integer
10Inhibition at 10.000 uM [ABHD6] (10μM**)Inhibition of endogenous mouse ABHD6 upon 10000 nM compound treatment as assessed by gel-based competitive ABPP.2Integer%
11Inhibition at 1.000 uM [ABHD6] (1μM**)Inhibition of endogenous mouse ABHD6 upon 1000 nM compound treatment as assessed by gel-based competitive ABPP.2Integer%
12Inhibition at 0.100 uM [ABHD6] (0.1μM**)Inhibition of endogenous mouse ABHD6 upon 100 nM compound treatment as assessed by gel-based competitive ABPP.2Integer%
13Inhibition at 0.010 uM [ABHD6] (0.01μM**)Inhibition of endogenous mouse ABHD6 upon 10 nM compound treatment as assessed by gel-based competitive ABPP.2Integer%
14Inhibition at 0.001 uM [ABHD6] (0.001μM**)Inhibition of endogenous mouse ABHD6 upon 1 nM compound treatment as assessed by gel-based competitive ABPP.2Integer%

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R01 DA025285

Classification
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