C646 is an inhibitor of histone acetyltransferase p300 (Ki=400 nM), which is competitive and selective, with less effect on other acetyltransferases. C646 acts as an apoptosis inducer and radiosensitizer.

p300/CBP:400 nM(Ki)

方法：静止 C3H10T1/2 小鼠成纤维细胞 SH-SY5Y 用 C646 (25 µM) 处理 1-3 h，加入 TSA (33 nM) 进行最后 30 min 的孵育，使用 Western Blot 检测靶点蛋白表达水平。
结果：在对照细胞中，在测试的 1-3 h 时间过程中，通过 C646 处理，H3 和 H4 的基础乙酰化水平略有降低。使用 HDAC 抑制剂 TSA 进行 30 min 的处理，其产生增加的 H3 和 H4 乙酰化。在添加 TSA 之前用 C646 孵育，几乎完全抑制了这种可诱导的乙酰化。[1]
方法：前列腺癌细胞系 C3、LNCaP、Du145 和 LAPC-4 用 C646 (5-20 µmol/L) 处理 24 h，通过发光底物检测 caspase 3/7 活性。
结果：在 caspase 3/7 活性测定中，PC3 和 Du145，特别是 LNCaP，在用 10-20 µmol/L C646 处理后都显示出细胞凋亡的增加。有趣的是，LAPC-4 在最高浓度下仅显示出 caspase 3/7 活性的边际增加。[2]

方法：为检测抗炎活性，将 C646 (5-10 mg/kg) 腹腔注射给 DSS 诱导结肠炎的 C57BL/6 小鼠模型，每天一次，持续十天。
结果：C646 通过靶向 NLRP3 炎症小体，在 DSS 诱导的结肠炎小鼠中发挥抗炎作用。[3]

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, 80 μM EDTA, 40μg/ml BSA, 100 μM 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°C 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°C, 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.

Histone acetylation assays in mouse cells. C3H10T1/2 mouse fibroblasts are grown in DMEM with 10% FCS at 37°C with 6% CO2. Confluent cultures are rendered quiescent in DMEM with 0.5% FCS for 18-20 hr prior to treatment. Cells are treated with the following compounds: TSA (10 ng/ml [33 nM]), C646 (25 μM), C37 (25 μM). Antibodies are used at the following concentrations: total H3 (1:10000; ab7834; Abcam); H4K12ac (1:2500; 06-761; Upstate). Rabbit anti-H3K9ac (1:10000) antibodies are generated in-house. Histones are isolated from cells by acid extraction, separated by SDS and acid-urea polyacrylamide gel electrophoresis and analyzed by western blotting.(Only for Reference)