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

Assays

PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		DAGLb	2	1	sn1-specific diacylglycerol lipase beta [Mus musculus] [gi:31559956]		Taxonomy id: 10090 Gene id: 231871
2		ABHD6	2	1	monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]		Taxonomy id: 10090 Gene id: 66082

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§ Panel component ID.

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)

This assay was performed by the assay provider with powder samples of synthetic compounds.

Grant Number: 1 R01 DA025285