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BioAssay: AID 2171

Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).

Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). ..more
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 Tested Compounds
 Tested Compounds
All(1514)
 
 
Active(1068)
 
 
Inactive(446)
 
 
 Tested Substances
 Tested Substances
All(1514)
 
 
Active(1068)
 
 
Inactive(446)
 
 
AID: 2171
Data Source: The Scripps Research Institute Molecular Screening Center (PME1_INH_FP_1536_3X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-12-03
Modify Date: 2010-06-15

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 1068
Depositor Specified Assays
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AIDNameTypeProbeComment
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).summary3 Summary AID.
2130Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening Primary screen (PME-1 inhibitors)
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Ben Cravatt, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R01 CA132630 Fast Track
Grant Proposal PI: Ben Cravatt, TSRI
External Assay ID: PME1_INH_FP_1536_3X%INH

Name: Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).

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 (10).

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.
10. Kidd, D., Liu, Y., Cravatt, B. F. (2001). Profiling serine hydrolase activities in complex proteomes. Biochemistry 40, 4005-4015.
11. Leung, D., Hardouin, C., Boger, D. L., Cravatt, B. F. (2003). Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21, 687-691.
12. Liu, Y., Patricelli, M. P., Cravatt, B. F. (1999). Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96, 14694-14699.

Keywords:

PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2a, lysophospholipase, LYPLA1, LYPLA2, cancer, fluorescence polarization, activity-based protein profiling, ABPP, fluorophosphonate rhodamine, FP-Rh, antagonist, inhibitor, confirmation, high throughput screen, HTS, 1536, Scripps, Scripps Florida, The 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 a previous set of experiments entitled, "Fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1)" (AID 2130). This competitive activity-based protein profiling (ABPP) assay uses fluorescence polarization to investigate enzyme-substrate functional interactions based on active site-directed molecular probes (11, 12). In this assay a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family (12) is used to label PME-1 in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Omission of enzyme (which gives the same result as use of a catalytically-dead enzyme) will serve as a positive control. Compounds were tested in triplicate at a nominal concentration of 5.9 micromolar.

Protocol Summary:

Prior to the start of the assay, 4.0 microliters of assay buffer (0.01% Pluronic acid, 50 mM Tris HCl pH8.0, 150 mM NaCl, 1mM DTT) containing 1.25 micromolar of PME-1 protein were dispensed into 1536 microtiter plates. Next, 30 nL of test compound in DMSO or DMSO alone (0.59% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 degrees Celsius.

The assay was started by dispensing 1.0 microliter of 375 nM FP-Rh probe in assay buffer to all wells. Plates were centrifuged and after 45 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.

The percent inhibition for each compound was calculated as follows:

Percent inhibition = ( Test_Compound_mP - median_Negative_Control_mP ) / ( median_ Positive_Control_mP - median_Negative_Control_mP ) * 100

Where:

Test_Compound is defined as wells containing PME-1 in the presence of test compound.
Negative_Control is defined as wells containing PME-1 and DMSO.
Positive_Control is defined as wells containing no PME-1 protein.

Any compound that exhibited a percent inhibition greater than the hit cutoff calculated for the primary screen was
declared active.

The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.

The activity score range for active compounds is 100-10, for inactive 10-0.

List of Reagents:

Recombinant PME-1 enzyme (supplied by Assay Provider)
Substrate (FP-Rh probe) (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
1536-well plates (Greiner, part 789176)
DTT (Invitrogen, part 15508-013)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this AID.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition (5.9μM**)Normalized percent inhibition of the confirmation screen at a compound concentration of 5.9 micromolar.Float%
2Standard DeviationStandard deviation of the confirmation screen derived from the normalized percent activation of the triplicate data for each compound.Float

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

Data Table (Concise)
Classification
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