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BioAssay: AID 624077

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 mouse liver ABHD6 in vivo upon oral compound administration

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 mouse liver ABHD6 in vivo upon oral compound administration. ..more
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(5)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(5)
 
 
AID: 624077
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD6_INH_FLUO_1X%INH_INVIVO_LIVER_PO)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-04-11
Hold-until Date: 2012-10-12
Modify Date: 2012-10-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 5
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 dose response (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)
624468Late stage assay provider results from the probe development effort to identify inhibitors of diacylglycerol lipase, beta (DAGLb): LCMS-based biochemical dose response assayConfirmatory depositor-specified cross reference
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 depositor-specified cross reference
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: ABHD6_INH_FLUO_1X%INH_INVIVO_LIVER_PO

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 mouse liver ABHD6 in vivo upon oral compound administration.

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 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 -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, DAGL-a/B, 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, oral bioavailability, in vivo, 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 whether or not powder samples of test compounds are orally bioavailable. In this assay, test compounds are orally administered to mice. Mice are sacrificed, and their liver tissue harvested, homogenized, and the membrane fraction isolated and reacted with the activity-based probe HT-01. HT-01 bears a BODIPY fluorophore and urea triazole reactive group that selectively labels several serine hydrolases. This reagent is used instead of FP-Rh to enhance visualization of select targets, which is otherwise obscured by other serine hydrolases upon SDS-PAGE separation/visualization. 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 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:
Purpose-bred C57-black laboratory mice were administered test compound (10, 5, or 1 mg/kg, p.o.) or vehicle only (n=1 per group). After 4 hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and liver tissue was removed and snap frozen in liquid nitrogen. Tissue was homogenized and the membrane fraction isolated by centrifugation (45 min, 100 K x g) and adjusted to 1 mg/mL in DPBS. Aliquots (50 uL) were reacted with the activity-based probe HT-01 (1 uL of a 50x stock in DMSO, 1 uM final concentration) for 30 minutes at 37 C. The reactions were 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 was determined by measuring the integrated optical density of test compound bands relative to vehicle bands.
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 mouse liver membrane fraction treated with test compound.
High_Control is defined as mouse liver membrane fraction 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 at 10 mg/kg compound were considered active. Compounds with less than 50% inhibition at 10 mg/kg compound were considered inactive.
The reported PubChem Activity Score has been normalized to 100% observed inhibition at 10 mg/kg. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.
List of Reagents:
C57-black laboratory mice (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 BioAssay Depositor:
BAO: assay format: biochemical format: protein format: single protein format
BAO: bioassay specification: assay biosafety level: bsl1
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: bioassay specification: assay stage: secondary: counter screening
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: biological process target: regulation of molecular function
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
Assay Test Type: In vivo
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 10 mg/kgInhibition of ABHD6 in mouse liver upon oral administration of 10 mg/kg cpd as assessed by gel-based competitive ABPP.Integer%
2Inhibition at 5 mg/kgInhibition of ABHD6 in mouse liver upon oral administration of 5 mg/kg cpd as assessed by gel-based competitive ABPP.Integer%
3Inhibition at 1 mg/kgInhibition of ABHD6 in mouse liver upon oral administration of 1 mg/kg cpd as assessed by gel-based competitive ABPP.Integer%
Additional Information
Grant Number: 1 R01 DA025285

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