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BioAssay: AID 652257

Primary biochemical fluorescence polarization-based high throughput screening assay to identify inhibitors of protein arginine methyltransferase 1 (PRMT1)

Name: Primary biochemical fluorescence polarization-based high throughput screening assay to identify inhibitors of protein arginine methyltransferase 1 (PRMT1). ..more
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 Tested Compounds
 Tested Compounds
All(369939)
 
 
Active(4757)
 
 
Inactive(365186)
 
 
 Tested Substances
 Tested Substances
All(370276)
 
 
Active(4763)
 
 
Inactive(365513)
 
 
AID: 652257
Data Source: The Scripps Research Institute Molecular Screening Center (PRMT1_INH_FP_1536_1X%INH)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-04-02
Modify Date: 2013-04-04

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 4757
Related Experiments
AIDNameTypeComment
686951Summary of the probe development effort to identify inhibitors of protein arginine methyltransferase 1 (PRMT1)Summarydepositor-specified cross reference
687036Fluorescence polarization-based biochemical high throughput confirmation assay to identify inhibitors of protein arginine methyltransferase 1 (PRMT1)Screeningdepositor-specified cross reference
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Kerri Mowen, The Scripps Research Institute
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA034598-01
Grant Proposal PI: Kerri Mowen, The Scripps Research Institute
External Assay ID: PRMT1_INH_FP_1536_1X%INH

Name: Primary biochemical fluorescence polarization-based high throughput screening assay to identify inhibitors of protein arginine methyltransferase 1 (PRMT1).

Description:

Protein Arginine Methyltransferases (PRMTs) catalyze the addition of a methyl group from S- adenosylmethionine (SAM) to guanidino nitrogen atoms on arginine residues. Many biological processes are regulated by PRMTs, including transcription, DNA repair, RNA processing, and signal transduction (1-2). PRMT1 is the predominant arginine methyltransferase, accounting for > 85% of cellular PRMT activity. Aberrant PRMT1 activity has been associated with cardiovascular, malignant, infectious, and autoimmune disease, making it a viable therapeutic target for several indications (2-8). PRMT1 can modify and regulate several critical immunomodulatory proteins. Post-translational modifications within T cell receptor signaling cascades allow T lymphocytes to initiate a rapid and appropriate immune response to pathogens (9-12). Since there is every indication that PRMT1 (and other arginine methyltransferase family members) occupy key positions in signaling pathways of relevance to inflammatory and cell regulatory diseases, it is likely that a high-throughput assay for PRMT1-specific inhibition would be useful for new therapeutic lead identification. In this project, an activity-based maleimide probe that labels thiols will be used in a fluorescence polarization-based HTS assay to identify lead PRMT1 inhibitor compounds. Identification of specific PRMT inhibitors would provide us with important tools to probe the importance of PRMT activity in T helper cell function.

References:

1. Bedford, M. T. (2007) Arginine methylation at a glance, J Cell Sci 120, 4243-4246.
2. Bedford, M. T., and Richard, S. (2005) Arginine methylation an emerging regulator of protein function, Mol Cell 18, 263-272.
3. Le Romancer, M., Treilleux, I., Bouchekioua-Bouzaghou, K., Sentis, S., and Corbo, L. (2010) Methylation, a key step for nongenomic estrogen signaling in breast tumors, Steroids 75, 560-564.
4. Mathioudaki, K., Papadokostopoulou, A., Scorilas, A., Xynopoulos, D., Agnanti, N., and Talieri, M. (2008) The PRMT1 gene expression pattern in colon cancer, Br J Cancer 99, 2094-2099.
5. Mathioudaki, K., Scorilas, A., Ardavanis, A., Lymberi, P., Tsiambas, E., Devetzi, M., Apostolaki, A., and Talieri, M. (2011) Clinical evaluation of PRMT1 gene expression in breast cancer, Tumour Biol 32, 575-582.
6. Nicholson, T. B., Chen, T., and Richard, S. (2009) The physiological and pathophysiological role of PRMT1-mediated protein arginine methylation, Pharmacol Res 60, 466-474.
7. Savoia, C., Ebrahimian, T., Lemarie, C. A., Paradis, P., Iglarz, M., Amiri, F., Javeshgani, D., and Schiffrin, E. L. (2010) Countervailing vascular effects of rosiglitazone in high cardiovascular risk mice: role of oxidative stress and PRMT-1, Clin Sci (Lond) 118, 583-592.
7. Savoia, C., Ebrahimian, T., Lemarie, C. A., Paradis, P., Iglarz, M., Amiri, F., Javeshgani, D., and Schiffrin, E. L. (2010) Countervailing vascular effects of rosiglitazone in high cardiovascular risk mice: role of oxidative stress and PRMT-1, Clin Sci (Lond) 118, 583-592.
8. Scoumanne, A., and Chen, X. (2008) Protein methylation: a new mechanism of p53 tumor suppressor regulation, Histol Histopathol 23, 1143-1149.
9. Blanchet, F., Cardona, A., Letimier, F. A., Hershfield, M. S., and Acuto, O. (2005) CD28 costimulatory signal induces protein arginine methylation in T cells, J Exp Med 202, 371-377.
10. Covic, M., Hassa, P. O., Saccani, S., Buerki, C., Meier, N. I., Lombardi, C., Imhof, R., Bedford, M. T., Natoli, G., and Hottiger, M. O. (2005) Arginine methyltransferase CARM1 is a promoter-specific regulator of NF-kappaB-dependent gene expression, EMBO J 24, 85-96.
11. Infantino, S., Benz, B., Waldmann, T., Jung, M., Schneider, R., and Reth, M. (2010) Arginine methylation of the B cell antigen receptor promotes differentiation, J Exp Med 207, 711-719.
12. Mowen, K. A., Schurter, B. T., Fathman, J. W., David, M., and Glimcher, L. H. (2004) Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes, Mol Cell 15, 559-571.

Keywords:

PRUN, Protein Arginine Methyltransferases, PRMT1, PRMTs, S- adenosylmethionine (SAM), guanidino nitrogen atoms, arginine residues, transcription, DNA repair, RNA processing, signal transduction, predominant arginine, cardiovascular, malignant, infectious, autoimmune disease, immunomodulatory proteins, Post-translational modifications, T cell receptor signaling, T lymphocytes, fluorescence, polarization-based, T helper cell function, 1536, inhibit, decrease, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC, Molecular Libraries Probe Production Centers Network, MLPCN.
Protocol
Assay Overview:
The purpose of this assay is to identify compounds that act as inhibitors of protein arginine methyltransferase 1 (PRMT1). In this assay, His-tagged recombinant PRMT1 protein is incubated with test compounds and Alexa Fluor 488-maleimide activity-based probe. 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 PRMT1 inhibitors will prevent PRMT1-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a final nominal concentration of 2.79 uM.
Protocol Summary:
Prior to the start of the assay, 4 uL of assay buffer (100 mM HEPES pH 7.0, 100 mM NaCl and 0.05% Pluronic F-127) containing 0.55 uM of PRMT1 protein were dispensed into 1536 microtiter plates. Next, 14 nL of test compound in DMSO or DMSO alone (0.27% final concentration) were added to the appropriate wells and incubated for 30 minutes at 25 C.
The assay was started by dispensing 1.0 uL of 25 nM Alexa Fluor 488-maleimide in assay buffer to all wells. Plates were centrifuged and after 60 minutes of incubation at 25 C, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a FITC FP filter set and a FITC dichroic mirror (excitation = 480 nm, emission = 540 nm). Fluorescence polarization was read for 40 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Where:
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
%_Inhibition = 100 * ( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Where:
Low_Control is defined as the median of the wells containing test compounds.
Test_Compound is defined as wells containing PRMT1 protein in the presence of test compound and Alexa Fluor 488-maleimide.
High_Control is defined as wells containing DMSO, Alexa Fluor 488-maleimide but, no PRMT1 protein.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
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 100-6, and for inactive compounds 6-0.
List of Reagents:
PRMT1 protein (supplied by Assay Provider)
Alexa Fluor 488-maleimide probe (Invitrogen, part A10254)
HEPES (Sigma, part 83264)
NaCl (Sigma, part S6546)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Corning, part 7261)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. 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 by the MLSMR.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Inhibition at 2.79 uM (2.79μM**)Normalized percent inhibition of the primary screen at a compound concentration of 2.79 micromolar.Float%

** Test Concentration.
Additional Information
Grant Number: 1 R03 DA034598-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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