Late 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 situ
Name: Late 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 situ. ..more
BioActive Compounds: 4
Depositor Specified Assays
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_3XIC50_INSITU
Name: Late 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 situ.
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.
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.
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, abhydrolase domain containing protein 6, ABHD6, activity-based protein profiling, ABPP, gel-based, activity-based probe, HT-01, inhibitor, inhibition, dose response, IC50, in situ, Neuro-2A, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
§ Panel component ID.
The purpose of this assay is to determine the IC50 values of powder samples of test compounds for anti-target ABHD6 inhibition in a complex proteome lysate. In this assay, a fluorescently-labeled activity-based probe, HT-01, which selectively labels several serine hydrolases including ABHD6, is used to label ABHD6 in the presence of test compounds. 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 ABHD6 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel.
Cultured Neuro-2A murine neuroblastoma cells (5 mL DMEM medium with 10% FCS) were treated with test compound (1 uL of 5000x stock in DMSO) or DMSO only (1 uL) and incubated for 4 hours at 37 C. Cells were washed with DPBS (4x), harvested, and homogenized by sonication. The protein concentration was adjusted to 2 mg/mL with in Dulbecco's PBS (DPBS), and an aliquot (50 uL) was reacted with HT-01 (1 uL of a 50x stock in DMSO; 1 uM final concentration) for 30 minutes at 37 C. Samples were quenched with an equal volume of 2x SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the ABHD6 band relative to the DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (8-point 1:3 dilution series from 0.1 uM to 0.000033 uM (100 nM to 0.033 nM)). Note: IC50 values and inhibition of DAGLb at each test concentration are also reported.
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
Test_Compound is defined as DAGLb or ABHD6 treated with test compound.
High_Control is defined as DAGLb or ABHD6 treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
For each test compound, percent inhibition was plotted against the log of the compound concentration. A three parameter equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported. In the event that the highest test concentration did not result in at least 50% inhibition, the IC50 value is reported as being greater than the highest test concentration.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with an IC50 less than or equal to 50 nM were considered active. Compounds with an IC50 greater than 50 nM were considered inactive.
Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
ABHD6 Score: The PubChem Activity Score range for active compounds 100-1. There are no inactive compounds.
DAGLb Score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
Overall Outcome and Score:
Compounds active for ABHD6 were considered active regardless of the DAGLb outcome. Compounds inactive for ABHD6 were considered inactive regardless of the DAGLb outcome.
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. There are no inactive compounds.
List of Reagents:
Neuro-2A cells (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.
BAO: version: 1.4b1090
BAO: bioassay specification: assay stage: secondary: counter screening
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
* Activity Concentration. ** Test Concentration. § Panel component ID.