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: HELACYTOX_INH_LUMI_384_4XCC50

Name: Late 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 compounds.

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, cytotoxicity, viability, luminescence, dose response, counterscreen, Hela, inhibitor, cancer, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN

Assays

PID§		Name	Substance		Panel Targets	Description
			Active	Inactive
1		Serum-free Media		4
2		Fetal Calf Serum		4

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§ Panel component ID.

Assay Overview:

The purpose of this assay is to determine cytotoxicity of inhibitor compounds belonging to aza-beta lactam scaffold. In this assay, HeLa cells in either serum-free media (Assay 1) or media containing FCS (Assay 2) are incubated with test compounds, followed by determination of cell viability. The assay utilizes the CellTiter-Glo luminescent reagent to measure intracellular ATP in viable cells. Luciferase present in the reagent catalyzes the oxidation of beetle luciferin to oxyluciferin and light in the presence of cellular ATP. Well luminescence is directly proportional to ATP levels and cell viability. As designed, compounds that reduce cell viability will reduce ATP levels, luciferin oxidation and light production, resulting in decreased well luminescence. Compounds were tested in quadruplicate in a 7-point 1:5 dilution series starting at a nominal test concentration of 50 uM.

Protocol Summary:

This assay was started by dispensing HeLa cells in RPMI media (15 uL, 5 x 10E3 cells/well) into a 384-well plate. Both serum-free media (Assay 1) and media supplemented with fetal calf serum (FCS) (Assay 2) were tested. Compound (5 uL of 0-200 uM in media containing 5% DMSO) was added to each well, giving final compound concentrations of 0-50 uM. Cells were incubated for 48hrs at 37 C in a humidified incubator and cell viability was determined by the CellTitre-Glo assay (Promega) according to manufacturer instructions.

The % cell viability for each well was then calculated as follows:

% Cell Viability = ( RFU_Test_Compound - MedianRFU_Low_Control ) / ( MedianRFU_High_Control - MedianRFU_Low_Control ) * 100

Where:

Test_Compound is defined as wells containing cells in the presence of test compound.
High_Control is defined as wells containing cells treated with media only (no compound).
Low_Control is defined as wells containing no cells (media only).

For each test compound, percent cell viability was plotted against the log of the compound concentration. The CC50 is reported as "> X uM" (where X = the highest concentration tested for which > 50% cell survival was observed).

PubChem Activity Outcome and Score:

Compounds with a CC50 value of less than 10 uM were considered active (cytotoxic). Compounds with a CC50 value greater than 10 uM were considered inactive (non-cytotoxic).

Any compound with a percent activity value > 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value < 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency of the compounds with fitted curves, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for inactive compounds is 100-0. There are no active compounds.

List of Reagents:

HeLa cells (provided by Assay Provider)
RPMI Media (CellGro 10-040-CV)
FCS (Omega Scientific, FB-01)
Cell Titer-Glo (Promega, part G7572)
384-well plates (Corning 3704)

This assay was performed by 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.

Grant Number: 1 R01 CA132630-01