Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).
Name: Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). ..more
Depositor Specified Assays
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Ben Cravatt, TSRI
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number 1 R01 CA132630 Fast Track
Grant Proposal PI: Ben Cravatt, TSRI
External Assay ID: PME1_INH_SUMMARY
Name: Summary of probe development efforts to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).
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 (10).
Summary of Probe Development Effort:
First PME-1 Probe (Sulfonyl Acrylonitrile Scaffold): Following the primary screen of the Maybridge library and counterscreening by gel-based competitive activity-based protein profiling (ABPP) in proteomes, a sulfonyl acrylonitrile class of inhibitor was identified for probe development. Following two rounds of SAR, compounds were tested for potency and selectivity in the presence of mouse brain soluble lysates in gel-based activity-based protein profiling (ABPP) assays, and for the ability to inhibit demethylation of PP2a by endogenous PME-1. This class of compound was shown to bind to PME-1 covalently by an ABPP gel filtration assay. Compound SID 87457340 was selected as a probe since it was the most potent and selective compound tested. It is ~20-fold more potent than the top initial hit (SID 26540970), and highly selective, as demonstrated by activity in the gel-based proteomic profiling assay. In addition, the probe compound SID 87457340 exhibits no cytotoxicity.
Second PME-1 Probe (Aza-beta-lactam Scaffold): Following uHTS primary, confirmation and counterscreening, and counterscreening by gel-based ABPP, an aza-beta-lactam class of inhibitor was identified for probe development. Compounds were tested for potency and selectivity in situ in HeLa cells and in the presence of HeLa soluble proteome in gel-based ABPP assays, and for the ability to inhibit demethylation of PP2a by endogenous PME-1. Compound SID 92709579 was identified as a highly potent and selective covalent inhibitor of PME-1 and selected as a probe. It has an IC50 of 10 nM, greater than 100-fold selectivity against potential serine hydrolase anti-targets, and exhibits no cytotoxicity (CC50 > 50 uM).
All AIDs that contain results associated with this project can be found in the "Related Bioassays" section of this Summary AID. Two probe reports have been submitted. The results of our probe development efforts can be found at http://mlpcn.florida.scripps.edu/index.php/probes/probe-reports. A probe report for SID 87457340 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML136. A probe report for SID 92709579 can be found in the Molecular Libraries Bookshelf (PubMed Books) (http://www.ncbi.nlm.nih.gov/books) under ML174.
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.
10. Kidd, D., Liu, Y., Cravatt, B. F. (2001). Profiling serine hydrolase activities in complex proteomes. Biochemistry 40, 4005-4015.
11. Leung, D., Hardouin, C., Boger, D. L., Cravatt, B. F. (2003). Discovering potent and selective reversible inhibitors of enzymes in complex proteomes. Nat. Biotechnol. 21, 687-691.
12. Liu, Y., Patricelli, M. P., Cravatt, B. F. (1999). Activity-based protein profiling: the serine hydrolases. Proc. Natl. Acad. Sci. U. S. A. 96, 14694-14699.
Summary, Summary AID, PME-1, protein phosphatase methylesterase 1, PPME-1, protein phosphatase 2a, PP2a, lysophospholipase, LYPLA1, LYPLA2, cancer, fluorescence polarization, activity-based protein profiling, ABPP, fluorophosphonate rhodamine, FP-Rh, antagonist, inhibitor, primary screen, high throughput screen, HTS, 1536, Scripps, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Bookshelf, Molecular Libraries Probe Production Centers Network, MLPCN.
§ Panel component ID.
Details of protocols, compound structures, and results from the original assays can be found in PubChem at the respective AIDs. Please see AID 2130 for all protocols performed in this probe development