Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds
Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds. ..more
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Benjamin Cravatt, TSRI
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R01 CA132630
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: NEURO2A_CYTOX_INH_ABSORB_6XCC50
Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: absorbance-based cell-based dose response assay to determine cytotoxicity of inhibitor compounds.
Protein phosphatase methylesterase-1 (PME-1)-mediated methylesterification is thought to control the binding of different subunits to protein phosphatase 2A (PP2A) (1), which, along with protein phosphatase 1 (PP1), is responsible for > 90% of all serine/threonine phosphatase activity (2). PME-1 has also been identified as a protector of sustained ERK pathway activity in malignant gliomas (3), suggesting a link between cancer progression and PME-1-regulated methylesterification. A fluorescence-polarization activity-based protein profiling (fluopol-ABPP) HTS assay for PME-1 inhibitor discovery (AIDs 2130 and 2171) unveiled a phenomenal class of potent and selective inhibitors, the aza-beta lactams (ABLs). During medicinal chemistry campaign to refine ABL inhibitors for PME-1 (See Probe Report for ML174 on the NCBI bookshelf http://www.ncbi.nlm.nih.gov/books/NBK47352/), we observed that one of the common anti-targets of several ABL members was the uncharacterized serine hydrolase abhydrolase domain containing protein 10 (ABHD10). We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored. A principle goal of post-genomic research is to elucidate the molecular and cellular roles of uncharacterized enzymes like ABHD10, work that requires selective chemical tools to inactivate enzyme activity in a controlled manner.
1. Wu, J., Tolstykh, T., Lee, J., Boyd, K., Stock, J. B., Broach, J. R. (2000). Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 19, 5672-5681.
2. Oliver, C. J., Shenolikar, S. (1998). Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 3, D961-972.
3. Puustinen, P., Junttila, M. R., Vanhatupa, S., Sablina, A. A., Hector, M. E., Teittinen, K., Raheem, O., Ketola, K., Lin, S., Kast, J., Haapasalo, H., Hahn, W. C., Westermarck, J. (2009). PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 69, 2870-2877.
late stage, late stage AID, assay provider, powders, abhdyrolase domain containing protein 10, ABHD10, uncharacterized, PME-1, protein phosphatase methylesterase 1, PPME-1, counterscreen, cytotoxicity, Neuro-2A, CC50, dose response, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this assay is to determine cytotoxicity of powder samples of synthetic inhibitor compounds belonging to the aza-beta lactam scaffold. In this assay, Neuro-2A murine neuroblastoma cells in either serum-free media or media containing fetal calf serum (FCS) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the WST-1 substrate that is converted into colorimetric formazan dye by the metabolic activity of viable cells. The amount of formed formazan directly correlates to the number of metabolically active cells in the culture. As designed, compounds that reduce cell viability will result in decreased absorbance of the dye.
This assay was started by seeding Neuro-2A murine neuroblastoma cells in DMEM medium (100 uL; 15,000 cells/well) into a 96-well plate. After 36 hours, the medium was removed and inhibitors were added in both serum-free media and media supplemented with fetal calf serum (FCS) (final compound concentration 0-10 uM in 100 uL total volume, 1% DMSO). Cells were incubated for 48 hours at 37 C in a humidified incubator and cell viability was determined using the WST-1 assay according to manufacturer instructions. CC50 values for inhibition of ABHD10 were determined from dose-response curves from six replicates at each inhibitor concentration (7-point 1:5 dilution series from 10 uM to 0.00064 uM).
The % surviving cells for each well was calculated as follows:
%_Surviving_Cells = ( ABS_Test_Compound - Median_ABS_Low_Control ) / ( Median_ABS_High_Control - Median_ABS_Low_Control ) * 100
Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).
In the event that the highest test concentration (10 uM) did not result in at least 50% cell death, the CC50 value is reported as being greater than the highest test concentration (10 uM). Otherwise, the CC50 value for each test compound was determined by plotting percent surviving cells 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), and the software-generated CC50 values are reported.
PubChem Activity Outcome and Score:
The following applies to each panel in this assay:
Compounds with CC50 less than or equal to 5 uM were considered active (cytotoxic). Compounds with CC50 greater than 5 uM were considered inactive (non-cytotoxic).
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
Serum-free media score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
Serum containing media score: The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
Overall Activity Outcome and Score:
Compounds with CC50 less than or equal to 5 uM in either panel were considered active (cytotoxic). Compounds with CC50 greater than 5 uM in both panels were considered inactive (non-cytotoxic).
The PubChem Activity Score range for inactive compounds is 0-0. There are no active compounds.
List of Reagents:
Neuro-2A murine cells (provided by Assay Provider)
DMEM Medium (CellGro 10-017-CV)
FCS (Omega Scientific, FB-01)
WST-1 reagent (Roche)
96-well plates (Corning)
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: 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 detail: binding reporter specification: interaction: protein-small molecule
BAO: detection technology: spectrophotometry: absorbance
* Activity Concentration. ** Test Concentration. § Panel component ID.