AlphaScreen Assay for Inhibitors of Human Jumonji Domain Containing 2E (JMJD2E)
The fine interplay among methylation states of several lysine residues on the tails of histone proteins is a major determinant of the transcriptional state of the associated DNA coding regions and is commonly referred to as the histone code. Histone lysine demethylases catalyze the removal of methyl groups from methylated lysine sidechains on histones H3 and H4, thus antagonizing the reactions more ..
BioActive Compounds: 4
The fine interplay among methylation states of several lysine residues on the tails of histone proteins is a major determinant of the transcriptional state of the associated DNA coding regions and is commonly referred to as the histone code. Histone lysine demethylases catalyze the removal of methyl groups from methylated lysine sidechains on histones H3 and H4, thus antagonizing the reactions catalyzed by histone lysine methyltransferases. The quest to define the biological roles of the multiple epigenetic modulator enzymes includes the identification and use of small molecules that selectively inhibit individual histone-modifying enzymes/enzyme subfamilies.
In search for novel inhibitors of JMJD2E demethylase, a member of the largest set of histone demethylases belonging to the Fe(II) and 2-oxoglutarate oxygenase (2OG) superfamily, we performed a quantitative high-throughput screen (Inglese 2006). See AID 2147 for details on the primary screen.
The assay here is a secondary AlphaScreen screen used to validate follow-up compounds.
Inglese, J., Auld, D., Jadhav, A., Johnson, R., Simeonov, A., Yasgar, A., Zheng, W. and Austin, C. Proc Natl Acad Sci USA 2006, 103, 11473-11478.
N. R. Rose, S. S. Ng, J. Mecinovic, B. M. Lienard, S. H. Bello, Z. Sun, M. A. McDonough, U. Oppermann and C. J. Schofield, J Med Chem, 2008, 51, 7053-7056.
Sakurai, M., Rose, N., Schultz, L., Quinn, A., Jadhav, A., Ng, S., Oppermann, U., Schofield, C.J. and Simeonov, A. Mol BioSystems 2009, in press.
S. Michael, D. Auld, C. Klumpp, A. Jadhav, W. Zheng, N. Thorne, C. Austin, J. Inglese and A. Simeonov, Assay Drug Dev Technol, 2008, 6, 637-657.
Assay Provider: Structural Genomics Consortium [SGC]
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Assay Buffer: 50 mM HEPES pH7.5, 0.01% Tween-20, 0.1% BSA (Cohn Fraction V; Sigma A7030)
Antibody: Abcam Ab1220, Anti-H3K9Me2
Final concentration of components in enzyme reaction:
JMJD2E 5 nM, Peptide 30 nM, 100 mM L-Ascorbic Acid, 1 mM Ferrous Ammonium Sulphate, 10 mM 2-OG, DMSO 0.1%
Alphascreen beads made up in the dark in assay buffer and pre-incubated for 1 hour in the dark.
Compounds counter-screened against 10 nM biotin-H3K9Me2 to check for interference of alphascreen chemistry.
Step 1. Compount Stock = 100mM
Step 2. Dilute compound to 1 mM in 50 mM HEPES pH7.5 + 0.01 % Tween-20 (final DMSO = 1%)
Step 3. Serial dilution (1:3 using 50 mM HEPES pH7.5 + 0.01 % Tween-20 containing 1% DMSO)
Step 4. Addition of 1 ul of compound per well (triplicates for each concentration)
Step 5. Addition of 5 ul per well of assay buffer containing 10 nM JMJD2E, 200 uM L-Ascorbic Acid, 2 uM Ferrous Ammonium Sulphate
Step 6. Incubate for 15 minutes at room temperature
Step 7. Addition of 4 ul assay buffer containing 75 nM Biotin-H3K9Me3, 25 uM 2-OG
Step 8. Incubate for 25 minutes at room temperature
Step 9. Addition of 5 ul of assay buffer containing 30 mM EDTA
Step 10. Addition of 5 ul Alphascreen Beads (0.08 mg / ml + 8 nM Ab) leave for 1 hour at room temperature
Step 11. Read plate on BMG Labtech Pherastar FS
Keywords: JMJD2, JMJD2E, Human 2-Oxoglutarate Oxygenase, qHTS, AlphaScreen
Compounds were ranked by taking the floor of the -Log_IC50*10. Compounds were 'active' if Potency < 100uM. If compound showed dose-dependent effect but had Potency > 100uM, it was considered 'inconclusive'. All other compounds were considered 'inactive'.
* Activity Concentration.
Data Table (Concise)