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BioAssay: AID 602320

Late 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 vitro

Name: Late 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 vitro. ..more
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
AID: 602320
Data Source: The Scripps Research Institute Molecular Screening Center (DAGLB_INH_FLUO_3XIC50_INVITRO)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-03-08
Hold-until Date: 2012-10-12
Modify Date: 2012-10-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 4
Depositor Specified Assays
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AIDNameTypeProbeComment
504411Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of human diacylglycerol lipase, beta (DAGLB)screening Primary screen (DAGLB inhibitors in singlicate)
504420Summary of the probe development efforts to identify inhibitors of human diacylglycerol lipase, beta (DAGLB)summary3 Summary (DAGLB inhibitors)
504445Fluorescence-based biochemical high throughput confirmation assay for inhibitors of human diacylglycerol lipase, beta (DAGLB)screening ABPP screen (DAGLB inhibitors in triplicate)
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
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
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
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
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
624468Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assayconfirmatory
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
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_3XIC50_INVITRO

Name: Late 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 vitro.

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, counterscreen, diacylglycerol lipase, diacylglycerol lipase-beta, DAGL, DAGL-beta, DAGLB, hydrolase, serine hydrolase, appetite, pain, sensation, memory, addiction, activity-based protein profiling, ABPP, gel-based, activity-based probe, HT-01, inhibitor, inhibition, dose response, IC50, 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 values of powder samples of test compounds for DAGLb inhibition in a complex proteome lysate. In this assay, a fluorescently-labeled activity-based probe, HT-01, which selectively labels several serine hydrolases including DAGLb, is used to label DAGLb in the presence of test compounds. 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 DAGLb 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 transiently transfected 293T Hek cells overexpressing mouse DAGLb (25 uL of 0.3 mg/mL) in Dulbecco's PBS (DPBS) was treated with test compound (0.5 uL of a 50x stock in DMSO) 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 activity remaining for DAGLb was determined by measuring the integrated optical density of the individual protein bands relative to the DMSO-only (no compound) control. IC50 values for inhibition of DAGLb were determined from dose-response curves from three replicates at each inhibitor concentration (10 uM and 8-point 1:3 dilution series from 1 uM to 0.00033 uM).

The percent inhibition for each compound was calculated as follows:

%_Inhibition = ( 1 -( IOD_Test_Compound - Median_IOD_Low_Control ) / ( Median_IOD_High_Control - Median_IOD_Low_Control ) ) * 100

Where:

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

For each test compound, percent inhibition 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 less than or equal to 0.5 uM were considered active. Compounds with an IC50 greater than 0.5 were considered inactive.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.

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 100-1. There are no inactive compounds.

List of Reagents:

293T Hek cells overexpressing mouse DAGLb (Open Biosystems Accession BC016105; 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
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate 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: fluorescence: fluorescence intensity

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
2Inhibition at 10 uM [1] (10μM**)The value for percent inhibition of endogenous mouse DAGLb at 10 uM compound concentration; replicate [1]Float%
3Inhibition at 1 uM [1] (1μM**)The value for percent inhibition of endogenous mouse DAGLb at 1 uM compound concentration; replicate [1]Float%
4Inhibition at 0.333 uM [1] (0.333μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.333 uM compound concentration; replicate [1]Float%
5Inhibition at 0.1 uM [1] (0.1μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.1 uM compound concentration; replicate [1]Float%
6Inhibition at 0.033 uM [1] (0.033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.033 uM compound concentration; replicate [1]Float%
7Inhibition at 0.01 uM [1] (0.01μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.01 uM compound concentration; replicate [1]Float%
8Inhibition at 0.0033 uM [1] (0.0033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.0033 uM compound concentration; replicate [1]Float%
9Inhibition at 0.001 uM [1] (0.001μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.001 uM compound concentration; replicate [1]Float%
10Inhibition at 0.00033 uM [1] (0.00033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.00033 uM compound concentration; replicate [1]Float%
11Inhibition at 10 uM [2] (10μM**)The value for percent inhibition of endogenous mouse DAGLb at 10 uM compound concentration; replicate [2]Float%
12Inhibition at 1 uM [2] (1μM**)The value for percent inhibition of endogenous mouse DAGLb at 1 uM compound concentration; replicate [2]Float%
13Inhibition at 0.333 uM [2] (0.333μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.333 uM compound concentration; replicate [2]Float%
14Inhibition at 0.1 uM [2] (0.1μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.1 uM compound concentration; replicate [2]Float%
15Inhibition at 0.033 uM [2] (0.033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.033 uM compound concentration; replicate [2]Float%
16Inhibition at 0.01 uM [2] (0.01μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.01 uM compound concentration; replicate [2]Float%
17Inhibition at 0.0033 uM [2] (0.0033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.0033 uM compound concentration; replicate [2]Float%
18Inhibition at 0.001 uM [2] (0.001μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.001 uM compound concentration; replicate [2]Float%
19Inhibition at 0.00033 uM [2] (0.00033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.00033 uM compound concentration; replicate [2]Float%
20Inhibition at 10 uM [3] (10μM**)The value for percent inhibition of endogenous mouse DAGLb at 10 uM compound concentration; replicate [3]Float%
21Inhibition at 1 uM [3] (1μM**)The value for percent inhibition of endogenous mouse DAGLb at 1 uM compound concentration; replicate [3]Float%
22Inhibition at 0.333 uM [3] (0.333μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.333 uM compound concentration; replicate [3]Float%
23Inhibition at 0.1 uM [3] (0.1μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.1 uM compound concentration; replicate [3]Float%
24Inhibition at 0.033 uM [3] (0.033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.033 uM compound concentration; replicate [3]Float%
25Inhibition at 0.01 uM [3] (0.01μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.01 uM compound concentration; replicate [3]Float%
26Inhibition at 0.0033 uM [3] (0.0033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.0033 uM compound concentration; replicate [3]Float%
27Inhibition at 0.001 uM [3] (0.001μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.001 uM compound concentration; replicate [3]Float%
28Inhibition at 0.00033 uM [3] (0.00033μM**)The value for percent inhibition of endogenous mouse DAGLb at 0.00033 uM compound concentration; replicate [3]Float%

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

Data Table (Concise)
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