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BioAssay: AID 463149

Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity. ..more
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 Tested Compounds
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Active(3)
 
 
Inactive(21)
 
 
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 Tested Substances
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Active(3)
 
 
Inactive(21)
 
 
AID: 463149
Data Source: The Scripps Research Institute Molecular Screening Center (PME1_INH_FLUO_GEL_1XINH_SEL)
BioAssay Type: Panel
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-08-26
Hold-until Date: 2011-06-09
Modify Date: 2011-06-10

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 3
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)
588796Late 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 2Confirmatory depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 same project related to Summary assay
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 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: PME1_INH_FLUO_GEL_1XINH_SEL

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity.

Description:

Reversible protein phosphorylation networks play essential roles in most cellular processes. While over 500 kinases catalyze protein phosphorylation, only two enzymes, PP1 and PP2A, are responsible for > 90% of all serine/ threonine phosphatase activity (1). Phosphatases, unlike kinases, achieve substrate specificity through complex subunit assembly and post-translational modifications rather than number. PP2A, for example, typically exists as heterotrimer with diverse subunits that may combinatorially make as many as 70 different holoenzyme assemblies (2). Mutations in several of these PP2A subunits have been identified in human cancers, suggesting that PP2A may act as a tumor suppressor (3). Adding further complexity, several residues of the catalytic subunit of PP2A can be reversibly phosphorylated, and the C-terminal leucine residue can be reversibly methylated (4,5). PME-1 is specifically responsible for demethylation of the carboxyl terminus (6).

Methylesterification is thought to control the binding of different subunits to PP2A, but little is known about physiological significance of this post-translational modification in vivo (7). Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas (8). In order to further elucidate the role of PP2A methylation in vivo, our lab has generated mice that lack PME-1 (PME-1 (-/-) mice) by targeted gene disruption (9). Unfortunately, PME-1 deletion resulted in perinatal lethality, underscoring the importance of PME-1 but hindering our biological studies. Biochemical elucidation of PME-1 would thus greatly benefit from the development of potent and selective chemical inhibitors.

References:

1. Oliver, C. J., Shenolikar, S. (1998). Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 3, D961-972.
2. Janssens, V., Goris, J. (2001). Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417-439.
3. Janssens, V., Goris, J., Van Hoof, C. (2005). PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 15, 34-41.
4. Chen, J., Martin, B. L., Brautigan, D. L. (1992). Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257, 1261-1264.
5. Favre, B., Zolnierowicz, S., Turowski, P., Hemmings, B. A. (1994). The catalytic subunit of protein phosphatase 2A is carboxyl-methylated in vivo. J. Biol. Chem. 269, 16311-16317.
6. Lee, J., Chen, Y., Tolstykh, T., Stock, J. (1996). A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. U. S. A. 93, 6043-6047.
7. 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.
8. 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.
9. Ortega-Gutierrez, S., Leung, D., Ficarro, S., Peters, E. C., Cravatt, B. F. (2008). Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS ONE 3, e2486.

Keywords:

late stage, late stage AID, assay provider, powders, PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2A, methylation, demethylation, counterscreen, selectivity, anti-targets, carboxylesterase, CES, fatty acid synthase, FAS, N-acylaminoacyl-peptide hydrolase, APEH, prolyl endopeptidase, PREP, ABHD10, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, Hela, inhibitor, selectivity, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN
Panel Information
Targets
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PME-1protein phosphatase methylesterase 1 [Homo sapiens] [gi:7706645]
Taxonomy id: 9606
Gene id: 51400
2Anti-Target CESCarboxylesterase 1 (monocyte/macrophage serine esterase 1) [Homo sapiens] [gi:82571745]
Taxonomy id: 9606
Gene id: 1066
3Anti-Target FASfatty acid synthase [Homo sapiens] [gi:119610151]
Taxonomy id: 9606
Gene id: 2194
4Anti-Target APEHN-acylaminoacyl-peptide hydrolase [Homo sapiens] [gi:12804773]
Taxonomy id: 9606
Gene id: 327
5Anti-Target PREPprolyl endopeptidase [Homo sapiens] [gi:119568811]
Taxonomy id: 9606
Gene id: 5550
6Anti-Target ABHD10Abhydrolase domain containing 10 [Homo sapiens] [gi:15778873]
Taxonomy id: 9606
Gene id: 55347

§ Panel component ID.
Protocol
Assay Overview:
The purpose of this assay is to determine whether powder samples of test compounds can inhibit PME-1 in a complex proteomic lysate and to estimate compound selectivity in an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction 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 (IOD) of the bands. As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-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:
HeLa soluble proteome (1 mg/mL in DPBS) was treated with 0.1 uM, 1 uM, or 20 uM test compound (1 uL of a 50X stock in DMSO). Test compounds were incubated for 30 min at 25 C (50 uL reaction volume). FP-Rh (1 uL of 50X stock in DMSO) was added to a final concentration of 1 uM. The reaction was incubated for 20 min at 25 C, quenched with 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 PME-1 band relative to a DMSO-only (no compound) control (see following equation). Observed anti-targets were carboxylesterase (CES), fatty acid synthase (FAS), N-acylaminoacyl-peptide hydrolase (APEH), prolyl endopeptidase (PREP), and ABHD10.
% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100
Where:
Test_Compound is defined as PME-1 or anti-target treated with test compound.
High_Control is defined as PME-1 or anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.
Fold selectivity is reported as:
[Conc_<50%_INH_Anti-target] / [Conc_>=50%_INH_PME-1]
Where:
[Conc_<50%_INH_Anti-target] is the test compound concentration at which less than 50% inhibition of the anti-target is observed.
[Conc_=50%_INH_PME-1] is the test compound concentration at which greater than or equal to 50% inhibition of PME-1 is observed.
If [Conc_<50%_INH_Anti-target] is 0.1 uM and [Conc_>=50%_INH_PME-1] is 1 uM, fold selectivity is reported as < 10. If the value for [Conc_>=50%_INH_PME-1] is greater than 1 uM, fold selectivity is not-determined.
PubChem Activity Outcome and Score:
Compounds with >= 50% inhibition at 0.1 uM and no anti-targets at 1 uM were considered active. Compounds with < 50% inhibition at 0.1 uM and/or one or more anti-targets at 1 uM were considered inactive.
Activity score was then ranked by the fold selectivity, largest fold selectivity receiving the highest activity scores. Inactive compounds were given a score of 0.
The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
List of Reagents:
HeLa soluble 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. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PME-1: Inhibition at 0.1 uM (0.1μM**)Inhibition of PME-1 in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.1protein phosphatase methylesterase 1 [Homo sapiens]Float%
2PME-1: Inhibition at 1 uM (1μM**)Inhibition of PME-1 in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.1Float%
3PME-1: Inhibition at 20 uM (20μM**)Inhibition of PME-1 in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.1Float%
4Anti-Target CES: Inhibition at 0.1 uM (0.1μM**)Inhibition of CES in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.2Carboxylesterase 1 (monocyte/macrophage serine esterase 1) [Homo sapiens]Float%
5Anti-Target CES: Qualifier at 1 uMActivity Qualifier identifies if the resultant percent inhibiton was determined manually to be less than or greater than its percent inhibition.2String
6Anti-Target CES: Inhibition at 1 uM (1μM**)Inhibition of CES in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.2Float%
7Anti-Target CES: Qualifier at 20 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.2String
8Anti-Target CES: Inhibition at 20 uM (20μM**)Inhibition of CES in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.2Float%
9Anti-Target FAS: Inhibition at 0.1 uM (0.1μM**)Inhibition of FAS in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.3fatty acid synthase [Homo sapiens]Float%
10Anti-Target FAS: Qualifier at 1 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.3String
11Anti-Target FAS: Inhibition at 1 uM (1μM**)Inhibition of FAS in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.3Float%
12Anti-Target FAS: Qualifier at 20 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.3String
13Anti-Target FAS: Inhibition at 20 uM (20μM**)Inhibition of FAS in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.3Float%
14Anti-Target APEH: Inhibition at 0.1 uM (0.1μM**)Inhibition of APEH in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.4N-acylaminoacyl-peptide hydrolase [Homo sapiens]Float%
15Anti-Target APEH: Inhibition at 1 uM (1μM**)Inhibition of APEH in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.4Float%
16Anti-Target APEH: Qualifier at 20 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.4String
17Anti-Target APEH: Inhibition at 20 uM (20μM**)Inhibition of APEH in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.4Float%
18Anti-Target PREP: Inhibition at 0.1 uM (0.1μM**)Inhibition of PREP in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.5prolyl endopeptidase [Homo sapiens]Float%
19Anti-Target PREP: Inhibition at 1 uM (1μM**)Inhibition of PREP in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.5Float%
20Anti-Target PREP: Qualifier at 20 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.5String
21Anti-Target PREP: Inhibition at 20 uM (20μM**)Inhibition of PREP in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.5Float%
22Anti-Target ABHD10: Inhibition at 0.1 uM (0.1μM**)Inhibition of ABHD10 in HeLa soluble proteome upon 0.1uM compound treatment as assessed by competitive ABPP.6Abhydrolase domain containing 10 [Homo sapiens]Float%
23Anti-Target ABHD10: Inhibition at 1 uM (1μM**)Inhibition of ABHD10 in HeLa soluble proteome upon 1uM compound treatment as assessed by competitive ABPP.6Float%
24Anti-Target ABHD10: Qualifier at 20 uMActivity Qualifier identifies if the resultant percent inhibition was determined manually to be less than or greater than its percent inhibition.6String
25Anti-Target ABHD10: Inhibition at 20 uM (20μM**)Inhibition of ABHD10 in HeLa soluble proteome upon 20uM compound treatment as assessed by competitive ABPP.6Float%
26Fold Selectivity QualiferActivity Qualifier identifies if the resultant fold selectivity was determined manually to be less than or greater than its listed fold selectivity.String
27Fold SelectivityThe ratio of the test compound concentration at which less than 50% inhibition of the anti-target is observed over the test compound concentration at which greater than or equal to 50% inhibition of PME-1 is observed.Float

** Test Concentration. § Panel component ID.
Additional Information
Grant Number: 1 R01 CA132630

Classification
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