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BioAssay: AID 588796

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 2. ..more
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Active(2)
 
 
AID: 588796
Data Source: The Scripps Research Institute Molecular Screening Center (ABHD10_INH_FLUO_GELBASEDABPP_3XIC50_SET2)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2011-11-17
Hold-until Date: 2012-05-31
Modify Date: 2012-05-31

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
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AIDNameTypeProbeComment
2130Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Summary3 depositor-specified cross reference: Summary (PME-1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Confirmation assay (PME-1 inhibitors)
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Counterscreen (LYPLA1 inhibitors)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Counterscreen (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).Screening depositor-specified cross reference: Counterscreen confirmation (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).Screening depositor-specified cross reference: Counterscreen confirmation (LYPLA1 inhibitors)
2291Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).Screening depositor-specified cross reference: Primary screen (PME-1 inhibitors, Maybridge Library)
2363Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Inhibition of PME-1-mediated demethylation of PP2aScreening depositor-specified cross reference: MOA assay (Demethylation of PP2a)
2365Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Luminescence-based counterscreen assay to identify cytotoxic compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HEC 293T)
2366Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50Confirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors)
2368Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) Gel Filtration AssayScreening depositor-specified cross reference: MOA assay (PME-1 inhibitors, gel filtration assay)
2369Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) InhibitionScreening depositor-specified cross reference: ABPP screen (PME-1 inhibitors)
2371Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50: Purified enzymeConfirmatory depositor-specified cross reference: ABPP dose response screen (PME-1 inhibitors, purified enzyme)
463090Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): LC-MS/MS assay to assess binding of compounds to active siteOther depositor-specified cross reference: MOA assay (PME-1 inhibitors, LC-MS assay)
463091Late stage assay provider results from the probe development effort to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1): luminescence-based biochemical dose response assay to determine cytotoxicity of inhibitor compoundsConfirmatory depositor-specified cross reference: Counterscreen (Cytotoxicity HeLa)
463124Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 2Confirmatory depositor-specified cross reference: ABPP profiling dose response (PME1 inhibitors in triplicate)
463130Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Gel-based Activity-Based Protein Profiling (ABPP) IC50 Set 1Confirmatory depositor-specified cross reference: ABPP profiling dose response (PME1 inhibitors in triplicate)
463131Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based inhibitionScreening depositor-specified cross reference: ABPP profiling (PME1 inhibitors in singlicate)
463132Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Inhibition of PME-1-mediated demethylation of PP2AScreening depositor-specified cross reference: MOA assay (PP2a demethylation)
463146Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): Fluorescence-based biochemical gel-based ABPPOther depositor-specified cross reference: ABPP profiling (PME1 inhibitors in singlicate)
463149Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivityOther depositor-specified cross reference: ABPP profiling (PME1 inhibition and selectivity in singlicate)
588835Late 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 assayOther depositor-specified cross reference
602468Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity among cysteine-reactive proteinsOther depositor-specified cross reference
602485Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 in vivoOther depositor-specified cross reference
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).Summary2 same project related to Summary assay
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).Summary1 same project related to Summary assay
588801Late 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) inhibition of ABHD10Confirmatory same project related to Summary assay
588802Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 1Confirmatory same project related to Summary assay
588803Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: LC-MS/MS-based cell-based ABPP-SILAC assayOther1 same project related to Summary assay
588804Late 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 compoundsConfirmatory same project related to Summary assay
588805Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC assayOther same project related to Summary assay
588806Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based cell-based gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10Other same project related to Summary assay
588807Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition and selectivity in a complex proteome for ABHD10Other same project related to Summary assay
Description:
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_SET2

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: fluorescence-based dose response biochemical gel-based competitive Activity-Based Protein Profiling (ABPP) inhibition of ABHD10 Set 2.

Description:

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.

References:

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.

Keywords:

late stage, late stage AID, assay provider, powders, dose response, abhdyrolase domain containing protein 10, ABHD10, uncharacterized, PME-1, protein phosphatase methylesterase 1, PPME-1, counterscreen, anti-targets, activity-based protein profiling, ABPP, gel-based, fluorophosphonate rhodamine, FP-Rh, inhibitor, selectivity, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Protocol
Assay Overview:
The purpose of this assay is to determine the IC50 values of powder samples of test compounds for ABHD10 inhibition in a complex proteome lysate. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe is used to label ABHD10 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 ABHD10 inhibitors will prevent enzyme-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.
Protocol Summary:
Mouse brain membrane proteome (1 mg/mL in DPBS) was incubated with DMSO or compound for 30 minutes at 37 C before the addition of FP-Rh at a final concentration of 1 uM in 50 uL total reaction volume. The reaction was incubated for 30 minutes at 25 C, 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 bands relative to DMSO only (no compound) control. IC50 values for inhibition of ABHD10 were determined from dose-response curves from three replicates at each inhibitor concentration (8-point 4-fold dilution series from 50 uM to 0.003 uM).
The % inhibition was then calculated as follows:
%_Inhibition = ( 1 - ( IOD_Test_Compound - Median_IOD_Low_Control ) / ( Median_IOD_High_Control - Median_IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as ABHD10 treated with test compound.
High_Control is defined as ABHD10 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.
PubChem Activity Outcome and Score:
Compounds with IC50 less than or equal to 0.500 uM were considered active. Compounds with IC50 greater than 0.500 uM were considered inactive.
Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.
The PubChem Activity Score range for active compounds is 100-1. There are no inactive compounds.
List of Reagents:
Mouse brain membrane proteome (provided by Assay Provider)
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)
Comment
This assay was performed by the assay provider with powder samples of synthetic compounds.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
BAO: assay format: biochemical format: protein format: protein complex format
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: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: fluorescence: fluorescence intensity
BAO: meta target detail: binding reporter specification: interaction: protein-small molecule
BAO: meta target: molecular target: protein target: enzyme: generic hydrolase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.FloatμM
2Inhibition at 50 uM [1] (50μM**)The value for percent inhibition of endogenous mouse ABHD10 at 50 uM compound concentration; replicate [1]Float%
3Inhibition at 50 uM [2] (50μM**)The value for percent inhibition of endogenous mouse ABHD10 at 50 uM compound concentration; replicate [2]Float%
4Inhibition at 50 uM [3] (50μM**)The value for percent inhibition of endogenous mouse ABHD10 at 50 uM compound concentration; replicate [3]Float%
5Inhibition at 12.5 uM [1] (12.5μM**)The value for percent inhibition of endogenous mouse ABHD10 at 12.5 uM compound concentration; replicate [1]Float%
6Inhibition at 12.5 uM [2] (12.5μM**)The value for percent inhibition of endogenous mouse ABHD10 at 12.5 uM compound concentration; replicate [2]Float%
7Inhibition at 12.5 uM [3] (12.5μM**)The value for percent inhibition of endogenous mouse ABHD10 at 12.5 uM compound concentration; replicate [3]Float%
8Inhibition at 3.125 uM [1] (3.125μM**)The value for percent inhibition of endogenous mouse ABHD10 at 3.125 uM compound concentration; replicate [1]Float%
9Inhibition at 3.125 uM [2] (3.125μM**)The value for percent inhibition of endogenous mouse ABHD10 at 3.125 uM compound concentration; replicate [2]Float%
10Inhibition at 3.125 uM [3] (3.125μM**)The value for percent inhibition of endogenous mouse ABHD10 at 3.125 uM compound concentration; replicate [3]Float%
11Inhibition at 0.781 uM [1] (0.781μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.781 uM compound concentration; replicate [1]Float%
12Inhibition at 0.781 uM [2] (0.781μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.781 uM compound concentration; replicate [2]Float%
13Inhibition at 0.781 uM [3] (0.781μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.781 uM compound concentration; replicate [3]Float%
14Inhibition at 0.195 uM [1] (0.195μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.195 uM compound concentration; replicate [1]Float%
15Inhibition at 0.195 uM [2] (0.195μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.195 uM compound concentration; replicate [2]Float%
16Inhibition at 0.195 uM [3] (0.195μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.195 uM compound concentration; replicate [3]Float%
17Inhibition at 0.049 uM [1] (0.049μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.049 uM compound concentration; replicate [1]Float%
18Inhibition at 0.049 uM [2] (0.049μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.049 uM compound concentration; replicate [2]Float%
19Inhibition at 0.049 uM [3] (0.049μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.049 uM compound concentration; replicate [3]Float%
20Inhibition at 0.012 uM [1] (0.012μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.012 uM compound concentration; replicate [1]Float%
21Inhibition at 0.012 uM [2] (0.012μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.012 uM compound concentration; replicate [2]Float%
22Inhibition at 0.012 uM [3] (0.012μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.012 uM compound concentration; replicate [3]Float%
23Inhibition at 0.003 uM [1] (0.003μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.003 uM compound concentration; replicate [1]Float%
24Inhibition at 0.003 uM [2] (0.003μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.003 uM compound concentration; replicate [2]Float%
25Inhibition at 0.003 uM [3] (0.003μM**)The value for percent inhibition of endogenous mouse ABHD10 at 0.003 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R01 CA132630

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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