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BioAssay: AID 463090

Late 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 site

Name: Late 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 site. ..more
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Active(1)
 
 
AID: 463090
Data Source: The Scripps Research Institute Molecular Screening Center (PME-1_INH_LCMS)
Depositor Category: NIH Molecular Libraries Probe Production Network, Assay Provider
BioAssay Version:
Deposit Date: 2010-08-09
Hold-until Date: 2011-06-09
Modify Date: 2011-06-10

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compound: 1
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)
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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 depositor-specified cross reference
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
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 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 CA132630-01
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME-1_INH_LCMS

Name: Late 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 site.

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. PMID: 8650216.
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. PMID: 11060018.
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. PMID: 19293187.
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. PMID: 18596935.


Keywords:

late stage, late stage AID, assay provider, powders, PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2a, methylation, demethylation, lysophospholipase, LYPLA1, LYPLA2, tumor suppressor, liquid chromatography-tandem mass spectrometry, LC-MS/MS, active site, trypsin, inhibitor, cancer, 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 assess the covalent nature of an inhibitor compound belonging to the aza-beta-lactam scaffold and determine whether or not it labels the active site serine of PME-1. In this assay, purified enzyme is reacted with inhibitor compound, digested with trypsin, and the resulting peptides are analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The resulting data are analyzed to identify sites of covalent labeling.

Protocol Summary:

Two aliquots (25 uL) of 50uM PME-1 in DPBS were prepared. To one aliquot was added inhibitor (0.5 uL of 10 mM in DMSO), giving a final concentration of 200 uM. To the second (control) aliquot was added DMSO (0.5 uL). Reactions were gently vortexed and incubated at room temperature for 30 min. To each reaction was added solid urea (50 mg), followed by freshly prepared aqueous ammonium bicarbonate (75 uL of 25 mM). The reactions were vortexed until the urea was dissolved. Final urea concentration was approximately 8 M. To each reaction was added freshly prepared TCEP (5 uL of 100 mM in water), and the reactions were incubated at 30 C for 30 min. To each reaction was then added freshly prepared IAA (10 uL of 100 mM in water), and the reactions were incubated for 30 min at room temperature in the dark. Aqueous ammonium bicarbonate (375 uL of 25 mM) was added to reduce the urea concentration to 2 M. To each reaction was added sequencing grade modified trypsin (1 ug), and reactions were incubated at 37 C for 12 hrs. The solvent was removed under reduced pressure in a SpeedVac, and peptides resuspended in ammonium bicarbonate containing 0.1% TFA.

An Agilent 1200 series quaternary HPLC pump and Thermo Scientific LTQ-Orbitrap mass spectrometer were used for sample analysis. A fraction (10 uL) of the protein digest for each sample was pressure-loaded onto a 100 micron fused-silica column (with a 5 micron in-house pulled tip) packed with 10 cm of Aqua C18 reversed-phase packing material. Chromatography was carried out using an increasing gradient of aqueous acetonitrile containing 0.1% formic acid over 125 minutes. Mass spectra were acquired in a data-dependent mode with dynamic exclusion enabled.

The MS/MS spectra generated for each run were searched against a human protein database concatenated to a reversed decoy database using Sequest. A static modification of +57.021 was specified cysteine, and a variable modification of +332.137 was specified for serine to account for possible probe labeling by 127A. The resulting peptide identifications were assembled into protein identifications using DTASelect, and filters were adjusted to maintain a false discovery rate (as determined by number of hits against the reversed database) of < 1%. Any modified peptides identified in the DMSO-treated sample were discarded as spurious hits.

PubChem Activity Outcome and Score:

Compounds observed to covalently modify the PME-1 active site serine were considered active. Compounds for which no covalent modification was observed were considered inactive.

The PubChem Activity Score is assigned a value of 100 for active compounds.

The PubChem Activity Score range for active compounds is 100-100. There are no inactive compounds.

List of Reagents:

PME-1 protein (provided by Assay Provider)
DPBS (CellGro 21-030-CV)
Urea (Fisher U15-3)
Ammonium bicarbonate (Acros, AC37093-0010)
TCEP (Tris[2-carboxyethyl]phosphine hydrochloride; Sigma C4706)
IAA (iodoacetamide; Sigma I1149)
Trypsin (Promega V5111)
TFA (trifluoroacetic acid; SigmaAldrich 302031)
Fused-silica (Agilent 160-2635-10)
Aqua C18 (Phenomenex 04A-4299)
Acetonitrile (Fisher A955-4)
Millipore-filtered Water
Formic acid (Fluka 06440)
Comment
This assay was performed in the laboratory of the Assay Provider with powder samples of compounds. 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
1Active site peptideTrypic peptide containing active site (* indicates modification of active site cysteine)String
2Active site covalently labeled?Whether the active site of PME-1 protein was covalently labeled by the compound, one of yes or noString
3Binding modeMode of binding of compound to active site of PME-1, one of covalent or noncovalentString
Additional Information
Grant Number: 1 R01 CA132630-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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