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BioAssay: AID 602345

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 macrophage DAGLb in vivo

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 macrophage DAGLb in vivo. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(3)
 
 
AID: 602345
Data Source: The Scripps Research Institute Molecular Screening Center (DAGLB_INH_FLUO_2X%INH_INVIVO)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2012-03-09
Hold-until Date: 2012-10-12
Modify Date: 2012-10-12

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 3
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_2X%INH_INVIVO

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 macrophage DAGLb in vivo.

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, hydrolase, serine hydrolase, appetite, pain, sensation, memory, addiction, activity-based protein profiling, ABPP, gel-based, activity-based probe, HT-01, inhibitor, inhibition, macrophages, 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 active in vivo. In this assay, test compounds are administered to mice. Mice are sacrificed, and macrophages 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 in addition to the standard serine hydrolase-specific probe fluorophosphonate-rhodamine (FP-Rh) to enhance visualization of select targets like DAGLb, 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, injected 4 days prior with thioglycollate, were administered test compound (20, 10, or 5 mg/kg in 18:1:1 saline:PEG300:EtOH vehicle solution, i.p.) or vehicle only (n=2 per group). After 4 hours, mice were humanely sacrificed (anesthetized with isoflurane and decapitated) and peritoneal macrophages were harvested and snap frozen in liquid nitrogen. Macrophages were homogenized and the membrane fraction isolated by centrifugation (45 min, 100K 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 % inhibition was then 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 at 5 mg/kg cpd were considered active. Compounds with less than 50% inhibition at 5 mg/kg cpd were considered inactive.

The reported PubChem Activity Score has been normalized to 100% observed inhibition at 5 mg/kg cpd. Negative % inhibition values are reported as activity score zero.

The PubChem Activity Score range for active compounds is 100-94. 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
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
1Inhibition at 20 mg/kg [1]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 20 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 1)Integer%
2Inhibition at 20 mg/kg [2]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 20 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 2)Integer%
3Inhibition at 10 mg/kg [1]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 10 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 1)Integer%
4Inhibition at 10 mg/kg [2]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 10 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 2)Integer%
5Inhibition at 5 mg/kg [1]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 5 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 1)Integer%
6Inhibition at 5 mg/kg [2]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 5 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 2)Integer%
7Inhibition at 1 mg/kg [1]Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. Percent administration of 1 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 1)Integer%
8Inhibition at 1 mg/kg [2]Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. Percent administration of 1 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 2)Integer%
9Inhibition at 0.5 mg/kg [1]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 0.5 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 1)Integer%
10Inhibition at 0.5 mg/kg [2]Percent Inhibition of DAGLb in mouse peritoneal macrophages upon i.p. administration of 0.5 mg/kg cpd as assessed by gel-based competitive ABPP. (Replicate 2)Integer%
Additional Information
Grant Number: 1 R01 DA025285

Data Table (Concise)
Classification
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