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_QFRET_1536_1X%INH PRUN

Name: Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of human diacylglycerol lipase, beta (DAGLB).

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, inhibitor, primary screen, uHTS, HTS, 1536, high throughput screen, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to identify compounds that inhibit the activity of human diacylglycerol lipase-beta (DAGL-beta). In this assay, compounds are preincubated with membranes derived from DAGL-beta-transfected HEK293T cells, followed by incubation with the fluorogenic lipase substrate EnzChek. Fluorescence is determined at a specific time point. As designed, compounds that act as DAGLB inhibitors will slow the rate of substrate hydrolysis, resulting in a decrease in well fluorescence. Compounds are tested in singlicate at a final nominal concentration of 5.17 uM.

Protocol Summary:

Prior to the start of the assay, DAGL-beta lysate was sonicated three times for 5 seconds, 1 minute between each pulse using a microtip. Then, 4.0 uL of Assay Buffer (50 mM HEPES pH7.2, 100 mM NaCl, 5 mM CaCl2, 0.1% Triton X-100, 10% DMSO and 0.5 mM DTT) containing 0.375 mg/mL of DAGL-beta membrane lysate were dispensed into 1536 microtiter plates. Next, 26 nL of test compound in DMSO or DMSO alone (0.52% final concentration) were added to the appropriate wells and incubated for 60 minutes at 25 C.

The assay was started by dispensing 1.0 uL of 5 uM EnzChek lipase substrate in Assay Buffer to all wells. Plates were centrifuged and incubated for 60 minutes at 25 C. Fluorescence was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a FITC filter set (excitation = 480 nm, emission = 540 nm) and a FITC dichroic mirror for 1 second.

The percent inhibition for each compound was calculated as follows:

%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )

Where:

High_Control is defined as wells containing no DAGLB protein.
Low_Control is defined as wells containing DAGLB and DMSO.
Test_Compound is defined as wells containing DAGLB in the presence of test compounds.

A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.

PubChem Activity Outcome and Score:

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

The PubChem Activity Score range for active compounds is 100-42, and for inactive compounds 42-0.

List of Reagents:

Membrane lysate derived from DAGL-beta-transfected HEK293T cells (supplied by Assay Provider)
EnzChek probe (Invitrogen part E33955)
HEPES (Cellgro, part 25-060-Cl)
NaCl (Sigma, part S6546)
Triton X-100 (Sigma, part T8787)
Calcium Chloride (Fisher, C79)
DMSO (Fisher, part NC9133613)
DTT (Invitrogen, part 15508-013)
Sonicator Ultrasonic Liquid Processor (Misonix, part S3000)
1536-well plates (Corning, part 7261)

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.

Grant Number: 1 R01 DA025285