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BioAssay: AID 2143

Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).

Name: Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Probe(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Probe(3)
 
 
Active(3)
 
 
AID: 2143
Data Source: The Scripps Research Institute Molecular Screening Center (PME1_INH_SUMMARY)
BioAssay Type: Panel, Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-11-16
Modify Date: 2012-06-04

Data Table ( Complete ):           Probe    Active    All
Target
BioActive Compounds: Chemical Probe: 3    Active: 3
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 Primary screen (PME-1 inhibitors)
2143Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).summary3 Summary (PME-1 inhibitors)
2171Fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening 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 Counterscreen screen (LYPLA1 inhibitors)
2177Counterscreen for PME1 inhibitors: fluorescence polarization-based primary biochemical high throughput screening assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen screen (LYPLA2 inhibitors)
2202Summary of probe development efforts to identify inhibitors of lysophospholipase 1 (LYPLA1).summary2 Summary (LYPLA1 inhibitors)
2203Summary of probe development efforts to identify inhibitors of lysophospholipase 2 (LYPLA2).summary1 Summary (LYPLA2 inhibitors)
2232Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of lysophospholipase 2 (LYPLA2).screening Counterscreen confirmation screen (LYPLA2 inhibitors)
2233Counterscreen for PME1 inhibitors: fluorescence polarization-based biochemical high throughput confirmation assay for inhibitors of lysophospholipase 1 (LYPLA1).screening Counterscreen confirmation screen (LYPLA1 inhibitors)
2291Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).screening Maybridge primary screen(PME-1 inhibitors)
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 Late stage (Inhibition of PME-1-mediated 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 Late stage counterscreen (Cytotoxicity)
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 Late stage gel-based ABPP dose response (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 Late stage gel-based ABPP gel filtration (PME-1 inhibitors)
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 Late stage gel-based ABPP inhibition (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 Late stage gel-based ABPP dose response purified enzyme (PME-1 inhibitors)
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 Late stage LC-MS/MS (PME-1 inhibitors)
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 Late stage dose response (Cytotoxicity)
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 Late stage dose response (PME-1 inhibitors)
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 Late stage gel-based ABPP dose response (PME-1 inhibitors)
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 Late stage fluorescence-base cell-based inhibition (PME-1 inhibitors)
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 Late stage (Inhibition of PME-1 mediated demethylation of PP2A)
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 Late stage fluorescence-based gel-based ABPP (PME-1 inhibitors)
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 Late stage fluorescence-based biochemical gel-based ABPP inhibition and selectivity (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 Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
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 Late stage dose response(ABPP inhibition of ABHD10 in triplicate)
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 Late stage dose response (ABPP inhibition of ABHD10 in triplicate)
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 Late stage results (LC-MS/MS-based cell-based ABPP-SILAC)
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 Late stage dose response assay (cytotoxicity in six replicates)
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 Late stage results (ABHD10 inhibitor LC-MS/MS-based cell-based ABPP-SILAC 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 Late stage results(ABPP inhibition of ABHD10)
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 Late stage assay (ABPP inhibition and selectivity in a complex proteome for ABHD10)
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 Late stage dose response (ABPP ABHD10 selectivity assay)
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 Late stage assay (cysteine-reactive proteins inhibition and selectivity)
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 Late stage assay (ABHD10 inhibitors in vivo)
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_SUMMARY

Name: Summary of probe development efforts to identify 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).

Summary of Probe Development Effort:

First PME-1 Probe (Sulfonyl Acrylonitrile Scaffold): Following the primary screen of the Maybridge library and counterscreening by gel-based competitive activity-based protein profiling (ABPP) in proteomes, a sulfonyl acrylonitrile class of inhibitor was identified for probe development. Following two rounds of SAR, compounds were tested for potency and selectivity in the presence of mouse brain soluble lysates in gel-based activity-based protein profiling (ABPP) assays, and for the ability to inhibit demethylation of PP2a by endogenous PME-1. This class of compound was shown to bind to PME-1 covalently by an ABPP gel filtration assay. Compound SID 87457340 was selected as a probe since it was the most potent and selective compound tested. It is ~20-fold more potent than the top initial hit (SID 26540970), and highly selective, as demonstrated by activity in the gel-based proteomic profiling assay. In addition, the probe compound SID 87457340 exhibits no cytotoxicity.

Second PME-1 Probe (Aza-beta-lactam Scaffold): Following uHTS primary, confirmation and counterscreening, and counterscreening by gel-based ABPP, an aza-beta-lactam class of inhibitor was identified for probe development. Compounds were tested for potency and selectivity in situ in HeLa cells and in the presence of HeLa soluble proteome in gel-based ABPP assays, and for the ability to inhibit demethylation of PP2a by endogenous PME-1. Compound SID 92709579 was identified as a highly potent and selective covalent inhibitor of PME-1 and selected as a probe. It has an IC50 of 10 nM, greater than 100-fold selectivity against potential serine hydrolase anti-targets, and exhibits no cytotoxicity (CC50 > 50 uM).

All AIDs that contain results associated with this project can be found in the "Related Bioassays" section of this Summary AID. Two probe reports have been submitted. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports. A probe report for SID 87457340 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML136. A probe report for SID 92709579 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML174.


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:

Summary, Summary AID, 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, primary screen, high throughput screen, HTS, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Bookshelf, Molecular Libraries Probe Production Centers Network, MLPCN.
Panel Information
AIDs
    Data Table(Active)    Data Table(All)Show more
PID§NameSubstancePanel TargetsDescriptionAdditional Information
ActiveInactive
1AID 2365
2AID 2366_1protein phosphatase methylesterase 1 [Homo sapiens] [gi:7706645]
Taxonomy id: 9606
Gene id: 51400
3AID 2366_2acylamino-acid-releasing enzyme [Homo sapiens] [gi:23510451]
Taxonomy id: 9606
Gene id: 327
4AID 2366_3
5AID 463124protein phosphatase methylesterase 1 [Homo sapiens] [gi:7706645]
Taxonomy id: 9606
Gene id: 51400
6AID 463091_1
7AID 463091_2
8AID 588801abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus] [gi:269784760]
Taxonomy id: 10090
Gene id: 213012
9AID 588802abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus] [gi:269784760]
Taxonomy id: 10090
Gene id: 213012
10AID 588804_1
11AID 588804_2
12AID 588835_1monoacylglycerol lipase ABHD6 [Mus musculus] [gi:31560264]
Taxonomy id: 10090
Gene id: 66082
13AID 588835_2prolyl endopeptidase [Mus musculus] [gi:6755152]
Taxonomy id: 10090
Gene id: 19072

§ Panel component ID.
Protocol
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs. Please see AID 2130 for all protocols performed in this probe development
effort.
Result Definitions
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TIDNameDescriptionPID§Panel TargetsHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML #Unique alphanumeric identifier assigned to a chemical probe molecule within the Molecular Libraries Probe Production Centers Network (MLPCN).String
2CC50 [AID 2365] The concentration at which 50 percent cytotoxicity is observed; (CC50) shown in micromolar.1FloatμM
3IC50 [AID 2366_1] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.2protein phosphatase methylesterase 1 [Homo sapiens]FloatμM
4Qualifier [AID 2366_2] 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.3acylamino-acid-releasing enzyme [Homo sapiens]String
5IC50 [AID 2366_2] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.3FloatμM
6Qualifier [AID 2366_3] 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.4String
7IC50 [AID 2366_3] The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.4FloatμM
8IC50 [AID 463124] The value for concentration at which 50% inhibition is observed; IC50 shown in micromolar.5protein phosphatase methylesterase 1 [Homo sapiens]FloatμM
9Qualifier [AID 463091_1] Activity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.6String
10CC50 [AID 463091_1] The value for concentration at which 50% of surviving cells are observed; CC50 shown in micromolar.6FloatμM
11Qualifier [AID 463091_2] Activity Qualifier identifies if the resultant data CC50 came from a fitted curve or was determined manually to be less than or greater than its listed CC50 concentration.7String
12CC50 [AID 463091_2] The value for concentration at which 50% of surviving cells are observed; CC50 shown in micromolar.7FloatμM
13IC50 [AID 588801] The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.8abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus]FloatμM
14IC50 [AID 588802] The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in micromolar.9abhydrolase domain-containing protein 10, mitochondrial precursor [Mus musculus]FloatμM
15Qualifier [AID 588804_1] Qualifier identifies if the resultant CC50 was determined manually to be greater than its listed CC50.10String
16CC50 [AID 588804_1] The value for the concentration at which 50% of surviving cells are observed in serum-free medium; CC50 shown in micromolar.10FloatμM
17Qualifier [AID 588804_2] Qualifier identifies if the resultant CC50 was determined manually to be greater than its listed CC50.11String
18CC50 [AID 588804_2] The value for the concentration at which 50% of surviving cells are observed in serum-containing medium; CC50 shown in micromolar.11FloatμM
19IC50 [AID 588835_1] The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in nanomolar.12monoacylglycerol lipase ABHD6 [Mus musculus]FloatμM
20IC50 [AID 588835_2] The average concentration at which 50 percent of the activity in the antagonist assay is observed; (IC50) shown in nanomolar.13prolyl endopeptidase [Mus musculus]FloatμM
Additional Information
Grant Number: 1 R01 CA132630

Classification
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