EML 425

Kinase experiment:

To explore the mechanisms of p300 inhibition by EML 425, reactions are performed. Each assay containing 5 nM p300, 3 μM Acetyl CoA, and 50 nM biotinylated H3 (1-21) peptide in 10 μL of assay buffer (50 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 1 mM DTT, 0.01% Tween-20, 0.01% BSA, 330 nM TSA) is incubated at room temperature for 15 min in a White opaque OptiPlate-384. Reactions are stopped by adding garcinol (final concentration 50 μM) and antiacetyl histone H3 lysine 9 (H3K9Ac) acceptor beads (final concentration 20 μg/mL). After 60 min of incubation at room temperature, 20 μg/mL final concentration of Alpha Streptavidin Donor beads are added in subdued light and incubated in the dark for 30 min at room temperature. Signals are read in Alpha mode with a Enspire plate reader[1].

Cell experiment:

For cell cycle analysis, 500 μL of U937 cells (2.5×105 cells/mL) are seeded in 24-well plastic plates and incubated with 100 μM EML 425 for 72 h. After this period of treatment, 500 μL of hypotonic buffer (33 mM sodium citrate, 0.1% Triton X-100, 50 μg/mL propidium iodide) is added to cell suspensions. Cells are analyzed with a FACScan flow cytometer and quantitative analysis of cell cycle distribution and hypodiploid nuclei is performed using ModFit LT Macintosh software. All the experiments are performed at least in triplicate[1].

References:

[1]. Milite C, et al. A novel cell-permeable, selective, and noncompetitive inhibitor of KAT3 histone acetyltransferases from a combined molecular pruning/classical isosterism approach. J Med Chem. 2015 Mar 26;58(6):2779-98.