Radioactive assay

IC50 values for the putative p300 HAT inhibitors are determined using the direct radioactive assay described above. Reactions are performed in 20 mM HEPES (pH 7.9), and contained 5 mM DTT, 80μM EDTA, 40μg/ml BSA, 100μM H4-15, and 5 nM p300. Putative inhibitors are added over a range of concentrations, with DMSO concentration kept constant (<5%). Reactions are incubated at 30℃ for 10 min, then initiated with addition of a 1:1 mixture of 12C-acetyl-CoA and 14C-acetyl-CoA to 20 mM. After 10 min at 30℃, reactions are quenched with 14% SDS (w/v). All concentrations are screened in duplicate. Gels are run, washed, dried, and exposed to a PhosphorImager plate, and production of Ac-H4-15 quantified to obtain IC50s.

Cell lines

RAW264.7 murine macrophages

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reacting condition

1, 5, 10, 15, 20, 25 or 30 μM for 16 hr; 15 or 30μM for 6 h, or at 30μM for 20 h

Applications

C646 decreased pro-inflammatory gene expression and NF-kB activity and inhibited histone deacetylases in RAW264.7 murine macrophages. Moreover, C646 decreased α-tubulin acetylation in RAW264.7 cells.

Animal models

C57BL/6 male mice model

Dosage form

2×0.75 μl injection volume in each case, 1.5 μg, administered over 2 min

Applications

Inhibition of p300 by C646 in the infralimbic prefrontal cortex (ILPFC) enhanced the formation of fear extinction memory.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

C646, a pyrazolone-containing small molecule, is a cell-permeable histone acetyltransferase (HAT) inhibitor that competitively and selectively inhibits the HAT activity of p300, a transcriptional co-activator involved in a variety of gene regulatory pathways and protein acetylation events, with the inhibition constant Ki value of 400 nM and the half maximal inhibition concentration IC50 value of 1.6 μM [1].

C646 binds to p300 as the Z-isomer forming hydrogen bonds between Thr1411, tYR1467, Trp1466 and Arg1410 on the side chains of p300 and oxygen atoms of C646 [1].

C646 is also capable of inhibiting a variety of p300 HAT mutants, including T1411A, Y1467F, W1466F and R1410A with IC50 values of 3.4 μM, 7 μM, 5 μM and 2.5 μM respectively [1].

Reference

References:
[1] Bowers EM, Yan G, Mukherjee C, Orry A, Wang L, Holbert MA, Crump NT, Hazzalin CA, Liszczak G, Yuan H, Larocca C, Saldanha SA, Abagyan R, Sun Y, Meyers DJ, Marmorstein R, Mahadevan LC, Alani RM, Cole PA. Virtual ligand screening of the p300/CBP histone acetyltransferase: identification of a selective small molecule inhibitor. Chem Biol. 2010 May 28;17(5):471-82. doi: 10.1016/j.chembiol.2010.03.006.