Data 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 Center Network (MLPCN)
Grant Proposal Number: 1 2 R01 CA087660-05 Fast Track
Grant Proposal PI: Benjamin Cravatt, TSRI
External Assay ID: PME-1_INH_FP_GEL_FILTRATION

Name: Late 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 Assay

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 (4).

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 (5). Recently, PME-1 has been identified as a protector of sustained ERK pathway activity in malignant gliomas 6. 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 (7). 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., Physiologic importance of protein phosphatase inhibitors. Front. Biosci. 1998, 3, D961-72.
2. Janssens, V.; Goris, J., Protein phosphatase 2A: a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 2001, 353 (Pt 3), 417-39.
3. Janssens, V.; Goris, J.; Van Hoof, C., PP2A: the expected tumor suppressor. Curr. Opin. Genet. Dev. 2005, 15 (1), 34-41.
4. Lee, J.; Chen, Y.; Tolstykh, T.; Stock, J., A specific protein carboxyl methylesterase that demethylates phosphoprotein phosphatase 2A in bovine brain. Proc. Natl. Acad. Sci. U. S. A. 1996, 93 (12), 6043-7.
5. Wu, J.; Tolstykh, T.; Lee, J.; Boyd, K.; Stock, J. B.; Broach, J. R., Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J. 2000, 19 (21), 5672-81.
6. 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., PME-1 protects extracellular signal-regulated kinase pathway activity from protein phosphatase 2A-mediated inactivation in human malignant glioma. Cancer Res. 2009, 69 (7), 2870-7.
7. Ortega-Gutierrez, S.; Leung, D.; Ficarro, S.; Peters, E. C.; Cravatt, B. F., Targeted disruption of the PME-1 gene causes loss of demethylated PP2A and perinatal lethality in mice. PLoS ONE 2008, 3 (7), e2486.

Keywords:

Late stage, reversible binding, gel filtration, Probes, PME-1, protein phosphatase methylesterase 1, PPME-1, cancer, fluorescence, fluorophosphonate rhodamine, FP-Rh, inhibitor, secondary, gel-based ABPP, Scripps, Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.

Assay Overview:

The purpose of this assay is to assess reversibility of binding of inhibitor compounds. In this assay, a fraction of the enzyme-inhibitor mixture is passaged over a Sephadex G-25M column before reaction with a serine hydrolase specific activity-based probe (FP-Rh). 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 of the bands. As designed, test compounds that act as irreversible PME-1 inhibitors will prevent PME-1-probe interactions after gel filtration, giving lower fluorescence intensity in the band in the gel. A compound's reversibility of inhibition of PME-1 is assessed by comparing the optical density of the bands with and without gel filtration.

Protocol Summary:

Recombinant PME-1 (2mM) in assay buffer was incubated with DMSO or inhibitor (20 mM) for 30 min at 25 degrees Celsius and each reaction was split into two fractions. One fraction was reacted directly with FP-Rh, and the other was passaged over a Sephadex G-25M column (GE Healthcare) and then reacted with FP-Rh at a final concentration of 75 nM in 50 ul total reaction volume. The reaction was incubated for 45 min at 25 degrees Celsius, quenched with 2x SDS-PAGE loading buffer, boiled for 5 min at 90 degrees Celsius, separated by SDS-PAGE and visualized in-gel.

Binding Mode:

A reversible inhibitor is defined as >50% recovery of labeling as measured by integrated optical intensity of the band following gel filtration; an irreversible inhibitor is defined as <10% recovery of labeling as measured by integrated optical intensity of the band following gel filtration; values between 10-50% are inconclusive.

PubChem Activity Score:

In this assay the PubChem Activity Score is assigned a value of 100 for probe compounds, 50 for active compounds, and 0 for inactive compounds.

List of Reagents:

Recombinant PME-1 protein (provided by Assay Provider)
FP-rhodamine (provided by Assay Provider)
Sodium Chloride (Fisher, part 980597)
1M Tris, pH 8.0 (Invitrogen, part T-3038)
Sephadex G-25 (GE Healthcare, part 17-0851-01)

This assay was performed in the laboratory of the Assay Provider with compounds ordered as powders. Probes were identified for this project. 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. Please also see Summary AID 2143. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports.html. One paper has been published detailing the emetine probe (7).

Grant Number: 1 2 R01 CA087660-05