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BioAssay: AID 2369

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

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) Inhibition ..more
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 Tested Compounds
 Tested Compounds
All(23)
 
 
Active(5)
 
 
Inactive(18)
 
 
 Tested Substances
 Tested Substances
All(23)
 
 
Active(5)
 
 
Inactive(18)
 
 
AID: 2369
Data Source: The Scripps Research Institute Molecular Screening Center (PME-1_INH_FP_GEL_%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity 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: 5
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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_%INH

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) Inhibition

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.
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. Percent inhibition of endogenously expressed PME-1 (compound at 20 uM) was determined.

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.

PubChem Activity Score:

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. The range for active compounds is 100-77, for inactive 66-0.

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. 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
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1% Inhibition (20μM**)Normalized percent inhibition of the primary screen at a compound concentration of 20 micromolarFloat%

** Test Concentration.
Additional Information
Grant Number: 1 2 R01 CA087660-05

Data Table (Concise)
Classification
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