Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1).
Name: Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase Methylesterase 1 (PME-1). ..more
BioActive Compounds: 73
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_FP_384_1X%INH Maybridge
Name: Fluorescence polarization-based Maybridge primary biochemical high throughput screening assay 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.
1. McConkey, DJ and Zhu, K, Mechanisms of proteasome inhibitor action and resistance in cancer. Drug Resist Updat, 2008. 11(4-5): p. 164-79.
2. Reed, JC, Drug insight: cancer therapy strategies based on restoration of endogenous cell death mechanisms. Nat Clin Pract Oncol, 2006. 3(7): p. 388-98.
3. Cory, S and Adams, JM, Killing cancer cells by flipping the Bcl-2/Bax switch. Cancer Cell, 2005. 8(1): p. 5-6.
4. Petros, AM, Olejniczak, ET and Fesik, SW, Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta, 2004. 1644(2-3): p. 83-94.
5. Redzepovic, J, Weinmann, G, Ott, I and Gust, R, Current trends in multiple myeloma management. J Int Med Res, 2008. 36(3): p. 371-86.
6. Derenne, S, Monia, B, Dean, NM, Taylor, JK, Rapp, MJ, Harousseau, JL, Bataille, R and Amiot, M, Antisense strategy shows that MCL1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells. Blood, 2002. 100(1): p. 194-9.
7. Kozopas, KM, Yang, T, Buchan, HL, Zhou, P and Craig, RW, MCL1, a gene expressed in programmed myeloid cell differentiation, has sequence similarity to BCL2. Proc Natl Acad Sci U S A, 1993. 90(8): p. 3516-20.
8. Opferman, JT, Iwasaki, H, Ong, CC, Suh, H, Mizuno, S, Akashi, K and Korsmeyer, SJ, Obligate role of anti-apoptotic MCL-1 in the survival of hematopoietic stem cells. Science, 2005. 307(5712): p. 1101-4.
9. Letai, A, Pharmacological manipulation of Bcl-2 family members to control cell death. J Clin Invest, 2005. 115(10): p. 2648-55.
10. Oltersdorf, T, Elmore, SW, Shoemaker, AR, Armstrong, RC, Augeri, DJ, Belli, BA, Bruncko, M, Deckwerth, TL, Dinges, J, Hajduk, PJ, Joseph, MK, Kitada, S, Korsmeyer, SJ, Kunzer, AR, Letai, A, Li, C, Mitten, MJ, Nettesheim, DG, Ng, S, Nimmer, PM, O'Connor, JM, Oleksijew, A, Petros, AM, Reed, JC, Shen, W, Tahir, SK, Thompson, CB, Tomaselli, KJ, Wang, B, Wendt, MD, Zhang, H, Fesik, SW and Rosenberg, SH, An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature, 2005. 435(7042): p. 677-81.
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.
Maybridge, 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, primary screen, high throughput screen, HTS, 1536, Scripps, Scripps La Jolla, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
The purpose of this assay is to identify compounds in the Maybridge collection that act as PME-1 inhibitors. This competitive activity-based protein profiling (ABPP) assay uses fluorescence polarization to investigate enzyme-substrate functional interactions based on active site-directed molecular probes (11, 12). In this assay a fluorophosphonate-rhodamine (FP-Rh) probe which broadly targets enzymes from the serine hydrolase family (12) is used to label PME-1 in the presence of test compounds. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as PME-1 inhibitors will prevent PME-1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Omission of enzyme (which gives the same result as use of a catalytically-dead enzyme) will serve as a positive control. Compounds were tested in singlicate at a nominal concentration of 9.0 micromolar.
Prior to the start of the assay, 10.0 microliters of Assay Buffer (0.1% Pluronic acid, 50 mM Tris HCl, 150 mM NaCl, pH 8.0) containing 1.1 micromolar of PME-1 protein were dispensed into 384 microtiter plates. Next, 50 nL of test compound in DMSO or DMSO alone (0.45% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 degrees Celsius.
The assay was started by dispensing 1.1 microliter of 750 nM FP-Rh probe in Assay Buffer to all wells. Plates were centrifuged and after 45 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm). Fluorescence polarization was read for 15 seconds for each polarization plane (parallel and perpendicular). The well fluorescence polarization value (mP) was obtained via the PerkinElmer Viewlux software.
The percent inhibition for each compound was calculated as follows:
Percent inhibition = ( Test_Compound_mP - median_Negative_Control_mP ) / ( median_Positive_Control_mP - median_Negative_Control_mP ) * 100
Test_Compound is defined as wells containing PME-1 in the presence of test compound.
Negative_Control is defined as wells containing PME-1 and DMSO.
Positive_Control is defined as wells containing no PME-1 protein.
Any compound that exhibited greater than 30% inhibition was declared active in the primary screen.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The PubChem Activity Score range for active compounds is 100-20, for inactive 20-0.
List of Reagents:
Recombinant PME-1 enzyme (supplied by Assay Provider)
Substrate (FP-Rh probe) (supplied by Assay Provider)
Tris HCl (Sigma, part T3038)
NaCl (Sigma, part S6546)
Pluronic acid (Invitrogen, part P6866)
384-well plates (Greiner, part 784076)
All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)