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BioAssay: AID 2366

Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50

Name: Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50 ..more
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 Tested Compounds
 Tested Compounds
All(26)
 
 
Active(10)
 
 
Inactive(16)
 
 
 Tested Substances
 Tested Substances
All(26)
 
 
Active(10)
 
 
Inactive(16)
 
 
AID: 2366
Data Source: The Scripps Research Institute Molecular Screening Center (PME-1_INH_FP_GEL_3XIC50)
BioAssay Type: Panel, Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-16
Hold-until Date: 2010-09-01
Modify Date: 2010-09-01

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 10
Depositor Specified Assays
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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 PME-1 inhibitors in singlate
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).summary3 Summary AID (PME-1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening PME-1 inhibitors in triplicate
2174Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 1 (LYPLA1).screening LYPLA1 inhibitors in singlate
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening LYPLA2 inhibitors in singlate
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening LYPLA2 inhibitors in triplicate
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening LYPLA1 inhibitors in triplicate
2291Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening PME-1 inhibitors in singlate
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 siteother1
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Description:
Data 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 Center Network (MLPCN)
Grant Proposal Number: 1 2 R01 CA087660-05 Fast Track
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME-1_INH_FP_GEL_3XIC50

Name: Late stage results from the probe development effort to identify inhibitors of the protein methylesterase PME-1: Gel-based Activity-Based Protein Profiling (ABPP) IC50

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 (4).

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 (5). Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas 6. 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 (7). 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., Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 1998, 3, D961-72.
2. Janssens, V.; Goris, J., Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 2001, 353 (Pt 3), 417-39.
3. Janssens, V.; Goris, J.; Van Hoof, C., PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005, 15 (1), 34-41.
4. Lee, J.; Chen, Y.; Tolstykh, T.; Stock, J., A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (12), 6043-7.
5. Wu, J.; Tolstykh, T.; Lee, J.; Boyd, K.; Stock, J. B.; Broach, J. R., Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 2000, 19 (21), 5672-81.
6. 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., PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 2009, 69 (7), 2870-7.
7. Ortega-Gutierrez, S.; Leung, D.; Ficarro, S.; Peters, E. C.; Cravatt, B. F., Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS ONE 2008, 3 (7), e2486.

Keywords:

Late stage, Probes, PME-1, protein phosphatase methylesterase 1, PPME-1, cancer, fluorescence, fluorophosphonate rhodamine, FP-Rh, inhibitor, confirmation, gel-based ABPP, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Panel Information
Assays
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1PME-1 Assay1016protein phosphatase methylesterase 1 [Homo sapiens] [gi:7706645]
Inhibition of endogenously expressed PME-1Taxonomy id: 9606
Gene id: 51400
2APEH Assay6N-acylaminoacyl-peptide hydrolase [Homo sapiens] [gi:23510451]
Inhibition of N-acylaminoacyl-peptide hydrolase (APEH) (anti-target)Taxonomy id: 9606
Gene id: 327
3ABPP Assay6Inhibition of more than 30 other serine hydrolases (anti-target)

§ Panel component ID.
Protocol
Assay Overview:

The purpose of this assay is to confirm activity of compounds identified as active in the uHTS primary (AID 2130) and confirmation screens (AID 2171). In this assay, a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family is used to label PME-1 in mouse brain soluble lysates 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 of the bands. As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, giving lower fluorescence intensity in the band in the gel.

Protocol Summary:

A preparation of mouse brain cytosol (2mg/mL) was incubated with DMSO or compound (20 mM) for 30 min at 25 degrees Celsius before the addition of FP-rhodamine at a final concentration of 75 nM in 50 ul total reaction volume. The reaction was incubated for 45 min at 25 degrees Celsius, quenched with 2x SDS-PAGE loading buffer, boiled for 5 min at 90 degrees Celsius, separated by SDS-PAGE and visualized in-gel. The percentage activity remaining was determined by measuring the integrated optical density of the bands. IC50 values for inhibition of endogenously expressed PME-1 (Assay 1), N-acylaminoacyl-peptide hydrolase (APEH) (anti-target) (Assay 2), and more than 30 other serine hydrolases (anti-target) (Assay 3) were determined from dose-response curves from three trials at each inhibitor concentration (0.1-100 uM).

PubChem Activity Score:

All compounds that were not tested in assays 2 and 3, for reasons of potency and selectivity, were assigned an activity score of 0. The activity score for the remaining compounds was then normalized over their potency range.

The activity score range for active compounds is 100-0, for inactive 0-0.

List of Reagents:

Mouse brain cytosol (provided by Assay Provider)
FP-rhodamine (provided by Assay Provider)
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
Comment
This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders.

Compounds not tested in assays 2 and 3 were because they were less potent and less selective analogs, and unlikely to be good candidates for medicinal chemistry optimization.

Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs listed below. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PME-1 Assay (Qualifier)Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.1protein phosphatase methylesterase 1 [Homo sapiens]String
2PME-1 Assay (IC50)*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.1FloatμM
3PME-1 Assay (Outcome)The Assay outcome, one of Active, Inactive or Not Tested1Outcome
4APEH Assay (Qualifier)Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.2N-acylaminoacyl-peptide hydrolase [Homo sapiens]String
5APEH Assay (IC50)*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.2FloatμM
6APEH Assay (Outcome)The Assay outcome, one of Active, Inactive or Not Tested2Outcome
7ABPP Assay (Qualifier)Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.3String
8ABPP Assay (IC50)*The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.3FloatμM
9ABPP Assay (Outcome)The Assay outcome, one of Active, Inactive or Not Tested3Outcome
10Probe Molecule OutcomeIndicates if the molecule is identified as a probe or not. One of Probe, Active or Inactive.String

* Activity Concentration. § Panel component ID.
Additional Information
Grant Number: 1 2 R01 CA087660-05

Classification
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