Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) ABHD10 selectivity assay
Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) ABHD10 selectivity assay ..more
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: ABHD10_INH_FLUO_GELBASEDABPP_3XIC50_INSITU_SEL
Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) ABHD10 selectivity assay
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), 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 We have preliminary evidence that ABHD10 functions as a lipase in situ (unpublished); however is physiological substrates and biological role(s) have not yet been explored.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. PMID: 11060018.
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. PMID: 19293187.
late stage, late stage AID, assay provider, powders, abhdyrolase domain containing protein 10, ABHD10, abhdyrolase domain containing protein 6, ABHD6, prolyl endopeptidase, PREP, uncharacterized, PME-1, protein phosphatase methylesterase 1, PPME-1, counterscreen, activity-based protein profiling, ABPP, inhibition, IC50, dose response, in situ, Neuro-2A, fluorophosphonate rhodamine, FP-Rh, inhibitor, selectivity, anti-targets, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
In situ assays
§ Panel component ID.
The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD10 anti-target inhibition in situ. In this assay, cultured cells are incubated with test compound. Cells are harvested, homogenized, and reacted with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. 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 anti-target 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 in serum-free DMEM medium (1 mL total volume) were treated with DMSO or test compound (10 uL of a 100x stock in DMSO) for 2 hours at 37 degrees Celsius. Cells were harvested, washed with DPBS (1 mL), and resuspended in DPBS (1 mL). Centrifugation (1000 x g, 5 minutes) provided a cell pellet which was resuspended in DPBS (200 uL) and homogenized by sonication. The protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50x stock in DMSO) was added to a final concentration of 1 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 degrees Celsius, quenched with 4x 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 anti-target ABHD6 or PREP band relative to a DMSO-only (no compound) control. IC50 values were determined from dose-response curves from three replicates at each inhibitor concentration (10 uM, 1 uM, 250 nM, 50 nM, 10 nM, 5 nM, and 1 nM).
%_Inhibition = ( 1 - ( IOD_Test_Compound-MedianIOD_Low_Control ) / ( MedianIOD_High_Control-MedianIOD_Low_Control ) ) * 100
Test_Compound is defined as anti-target treated with test compound.
High_Control is defined as anti-target 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 four parameter variable slope equation describing a sigmoidal dose-response curve was then fitted using GraphPad Prism (GraphPad Software Inc). The software-generated IC50 values are reported.
ABHD6 and PREP Assay Outcomes:
Compounds with IC50 > 0.5 uM were considered inactive. There are no active compounds.
PubChem Activity Outcome:
Compounds that were "inactive" in both ABHD6 and PREP assays were considered "inactive". Compounds that were "active" in one or both ABHD6 and PREP assays were considered "active".
PubChem Activity Score:
The PubChem Activity Score is assigned a value of 50 for active compounds, and 0 for inactive compounds.
List of Reagents:
Neuro-2A murine cells (provided by Assay Provider)
FP-Rh (provided by the Assay Provider)
DMEM Medium (CellGro 10-017-CV)
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: selectivity
BAO: bioassay specification: assay biosafety level: bsl1
BAO: assay format: biochemical format: protein format: protein complex 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 detail: binding reporter specification: interaction: protein-small molecule
BAO: detection technology: fluorescence: fluorescence intensity
* Activity Concentration. ** Test Concentration. § Panel component ID.