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: PME-1_INH_FLUO_ABPP_INSITU

Name: Late stage assay provider results from the probe development effort to identify inhibitors of protein phosphatase methylesterase 1 (PME-1): fluorescence-based cell-based 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). Protein phosphatase methylesterase 1 (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, activity-based protein profiling, ABPP, gel-based ABPP, HeLa, SDS-PAGE, in situ, fluorescence, fluorophosphonate rhodamine, FP-Rh, inhibitor, Scripps, Scripps Florida, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to determine whether or not powder samples of test compounds can inhibit PME-1 activity in situ. In this assay, cultured HeLa cells are incubated with test compound. Cells are harvested and the soluble fraction is isolated and reacted 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.

Protocol Summary:

HeLa cells in media (5 mL total volume; supplemented with FCS) were treated with 500 nM test compound (5 uL of a 1000X stock in DMSO) for 1 hour at 37 C. Cells were harvested, washed 4 times with 10 mL DPBS, and homogenized by sonication in DPBS. The soluble fraction was isolated by centrifugation (100K x g, 45 min) and the protein concentration was adjusted to 1 mg/mL with DPBS. FP-Rh (1 uL of 50X stock in DMSO) was added to a final concentration of 1 uM in 50 uL total reaction volume. 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.

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

Where:

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

PubChem Activity Outcome and Score:

Compounds with >= 90% inhibition were considered active. Compounds with inhibition < 90% inhibition 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:

HeLa cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
DPBS (Cellgro 20-031-CV)
FP-Rh (provided by the Assay Provider)

This assay was performed by the assay provider with powder samples of 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 CA132630-01