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].

EML425 is a potent and selective CREB binding protein (CBP)/p300 inhibitor with IC50s of 2.9 and 1.1 μM, respectively.

EML 425 (EML425, Compound 7h) is a potent and selective reversible inhibitor of CBP/p300, noncompetitive versus both acetyl-CoA and a histone H3 peptide, and endows with good cell permeability. EML 425 inhibits both p300 and CBP (IC50 values of 2.9 and 1.1 μM, respectively) while being practically inactive against the enzymes general control non derepressible-5 (GCN5) and p300/CBP-associated factor (PCAF). EML 425 induces a marked and time-dependent reduction in the acetylation of lysine H4K5 and H3K9 in U937 cells. EML 425 is shown to be a reversible inhibitor, noncompetitive versus both acetyl-CoA and a histone H3 peptide, and able to bind both the free enzyme and the enzyme-substrate complex, even with unequal affinity constants. The best scoring docking poses suggest that the binding site for EML 425 is an alternative pocket lying near the substrate lysine binding groove and close to the acetylation site[1].

[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.