RGFP966 is an HDAC3 inhibitor (IC50: 0.08 μM) and does not affect other HDACs at concentrations up to 15 μM.

HDAC3:80 nM(cell free)

RGFP966 对HDAC3具有特异性抑制作用 （IC50：0.08 μM），在高至15 μM的浓度下，对其他HDAC无有效抑制 [1]。在LPS/IFNγ刺激的RAW 264.7巨噬细胞中，RGFP966处理未改变TNFα、iNOS和IL-10基因的表达，但显著降低了促炎症基因IL-1β、IL-6和IL-12b的表达 [2]。此外，RGFP966在CTCL细胞系中减缓了细胞增长，此现象与DNA损伤和S期进程受损相关的增加的细胞凋亡有关 [3]。

所有小鼠在经过可卡因条件性位置偏好(CPP)训练后，明显偏好于与可卡因相关联的环境。在无药物偏好测试后立即使用RGFP966（3或10 mg/kg，皮下注射）治疗，在第二次和第三次后测中显著减弱了CPP。其中，10 mg/kg的剂量显著快速降低了随后几天的CPP，而3 mg/kg的剂量则没有此效果[1]。RGFP966在10和25 mg/kg的剂量下，能改善旋转木马测试和开放场探索中的运动缺陷，并伴随着对纹状体体积的神经保护作用[4]。

Briefly, the respective human recombinant HDAC enzymes were incubated in the absence and/or in presence of various concentrations RGFP966 and a pro-fluorogenic substrate at room temperature for 60 min. Next, the deacetylation reaction was stopped by the addition of the HDAC Stop Solution (6 mg/ml trypsin, 0.3 mM SAHA) in all wells and the plate was incubated at 37 °C for 20 min. The release of the fluorescent 7-amino-4-methylcoumarin was monitored by measuring the fluorescence at λem = 460 nm and λex = 390 nm using a Synergy H1 plate reader. The fluorescence value of the background wells was subtracted from the fluorescence of the positive control, blank and inhibitor wells. Nonlinear regression was used to fit the data to the log(inhibitor) vs. response curve using GraphPad Prism [2].

To investigate the influence of the HDAC 3-selective inhibitor RGFP966 on cell viability, RAW 264.7 macrophages, HBE cells and hASM cells were seeded in 96-well plates. To obtain identical cell density at the start of the experiments, RAW 264.7 macrophages were seeded at 25,000 cells/cm2, HBE cells and hASM cells were seeded at 70% confluency (based on surface area) and were serum-starved for 24 h prior incubation with RGFP966. Shortly before incubation with RGFP966, the medium was replaced by 100 μl fresh (if appropriate serum free) culture medium. Incubations with LPS and IFNγ were performed as described for HDAC 1–3 downregulation by siRNA. After 20 h of incubation with RGFP966, 20 μl of CellTiter 96 AQueous One Solution reagent was added to each well and incubated at 37 °C for 1 h in the dark. The absorbance at 490 nm was measured using a Synergy H1 plate reader. LPS/IFNγ-stimulated cells without addition of RGFP966 were considered 100% [2].

Subthreshold training and a 24-h retention test for location-dependent object recognition memory (OLM) and novel object recognition memory (ORM) were performed as described previously. Mice received an injection of RGFP966 (3, 10, or 30 mg/kg s.c) or vehicle alone either 1 h before or immediately after a 3-min training seeion [1].