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BioAssay: AID 602299

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 overexpressed DAGLb in vitro set 2

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 overexpressed DAGLb in vitro set 2. ..more
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 Tested Compounds
 Tested Compounds
All(8)
 
 
Active(6)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(8)
 
 
Active(6)
 
 
Inactive(2)
 
 
AID: 602299
Data Source: The Scripps Research Institute Molecular Screening Center (DAGLB_INH_FLUO_1X%INH_OVEREXPRESSED_SET2)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-03-06
Hold-until Date: 2012-10-12
Modify Date: 2012-10-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 6
Depositor Specified Assays
Show more
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)
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_1X%INH_OVEREXPRESSED_SET2

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 overexpressed DAGLb in vitro set 2.

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:

Diacylglycerol lipase, Diacylglycerol lipase-beta, DAGL, DAGL-b, DAGL-beta, DAGLB, DAGL-B, hydrolase, serine hydrolase, appetite, pain, sensation, memory, addiction, fluorescence, HEK293T, membrane, lysate, EnzChek, fluorogenic lipase substrate, QFRET, inhibitor, confirmation, triplicate, uHTS, HTS, 1536, high throughput screen, Scripps, Scripps Florida, 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 whether test compounds can inhibit DAGLb in a gel-based competitive activity-based proteomic profiling (ABPP) assay. In this assay, the target enzyme DAGLb 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. 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; 10 uM 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 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 - 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.

PubChem Activity Outcome and Score:

Compounds with greater than or equal to 50% inhibition were considered active. Compounds with less than 50% inhibition were considered inactive.

reported PubChem Activity Score has been normalized to 100% observed inhibition. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-50, and for inactive compounds 39-0.

List of Reagents:

293T Hek cells overexpressing mouse DAGLb (Open Biosystems Accession BC016105; provided by Assay Provider)
FP-Rh (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
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 10 uM (10μM**)Inhibition of overexpressed mouse DAGLb upon 10 uM compound treatment as assessed by gel-based competitive ABPP.Integer%

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

Data Table (Concise)
Classification
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