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
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME1_INH_FLUO_GEL_1XINH_SEL

Name: Late stage assay provider results from the probe development effort to identify inhibitors of PME-1: Fluorescence-based biochemical gel-based ABPP inhibition and selectivity.

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.
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.

Keywords:

late stage, late stage AID, assay provider, powders, PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2A, methylation, demethylation, counterscreen, selectivity, anti-targets, carboxylesterase, CES, fatty acid synthase, FAS, N-acylaminoacyl-peptide hydrolase, APEH, prolyl endopeptidase, PREP, ABHD10, activity-based protein profiling, ABPP, gel-based ABPP, fluorophosphonate rhodamine, FP-Rh, Hela, inhibitor, selectivity, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Targets

Data Table(Active)    Data Table(All)

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PID§		Name	Substance		Panel Targets	Description	Additional Information
			Active	Inactive
1		PME-1			protein phosphatase methylesterase 1 [Homo sapiens] [gi:7706645]		Taxonomy id: 9606 Gene id: 51400
2		Anti-Target CES			Carboxylesterase 1 (monocyte/macrophage serine esterase 1) [Homo sapiens] [gi:82571745]		Taxonomy id: 9606 Gene id: 1066
3		Anti-Target FAS			fatty acid synthase [Homo sapiens] [gi:119610151]		Taxonomy id: 9606 Gene id: 2194
4		Anti-Target APEH			N-acylaminoacyl-peptide hydrolase [Homo sapiens] [gi:12804773]		Taxonomy id: 9606 Gene id: 327
5		Anti-Target PREP			prolyl endopeptidase [Homo sapiens] [gi:119568811]		Taxonomy id: 9606 Gene id: 5550
6		Anti-Target ABHD10			Abhydrolase domain containing 10 [Homo sapiens] [gi:15778873]		Taxonomy id: 9606 Gene id: 55347

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§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine whether powder samples of test compounds can inhibit PME-1 in a complex proteomic lysate and to estimate compound selectivity in an activity-based proteomic profiling (ABPP) assay. In this assay, a complex proteome is incubated with test compound followed by reaction with a rhodamine-conjugated fluorophosphonate (FP-Rh) activity-based probe. 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 (IOD) of the bands. As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Percent inhibition is calculated relative to a DMSO (no compound) control.

Protocol Summary:

HeLa soluble proteome (1 mg/mL in DPBS) was treated with 0.1 uM, 1 uM, or 20 uM test compound (1 uL of a 50X stock in DMSO). Test compounds were incubated for 30 min at 25 C (50 uL reaction volume). FP-Rh (1 uL of 50X stock in DMSO) was added to a final concentration of 1 uM. The reaction was incubated for 20 min at 25 C, quenched with 2X SDS-PAGE loading buffer, separated by SDS-PAGE and visualized by in-gel fluorescent scanning. The percentage activity remaining was determined by measuring the integrated optical density of the PME-1 band relative to a DMSO-only (no compound) control (see following equation). Observed anti-targets were carboxylesterase (CES), fatty acid synthase (FAS), N-acylaminoacyl-peptide hydrolase (APEH), prolyl endopeptidase (PREP), and ABHD10.

% Inhibition = ( 1 - ( IOD_Test_Compound - IOD_Low_Control ) / ( IOD_High_Control - IOD_Low_Control ) ) * 100

Where:

Test_Compound is defined as PME-1 or anti-target treated with test compound.
High_Control is defined as PME-1 or anti-target treated with DMSO only (no compound).
Low_Control is defined as background in a blank region of the gel.

Fold selectivity is reported as:

[Conc_<50%_INH_Anti-target] / [Conc_>=50%_INH_PME-1]

Where:

[Conc_<50%_INH_Anti-target] is the test compound concentration at which less than 50% inhibition of the anti-target is observed.
[Conc_=50%_INH_PME-1] is the test compound concentration at which greater than or equal to 50% inhibition of PME-1 is observed.

If [Conc_<50%_INH_Anti-target] is 0.1 uM and [Conc_>=50%_INH_PME-1] is 1 uM, fold selectivity is reported as < 10. If the value for [Conc_>=50%_INH_PME-1] is greater than 1 uM, fold selectivity is not-determined.

PubChem Activity Outcome and Score:

Compounds with >= 50% inhibition at 0.1 uM and no anti-targets at 1 uM were considered active. Compounds with < 50% inhibition at 0.1 uM and/or one or more anti-targets at 1 uM were considered inactive.

Activity score was then ranked by the fold selectivity, largest fold selectivity receiving the highest activity scores. Inactive compounds were given a score of 0.

The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.

List of Reagents:

HeLa soluble proteome (provided by Assay Provider)
FP-Rh (provided by the Assay Provider)
DPBS (Cellgro 20-031-CV)

This assay was performed by the assay provider with powder samples of synthetic compounds. Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDS listed in the Related Bioassays section of this AID.

Grant Number: 1 R01 CA132630