Deacetylation assays

Deacetylation assays were based on the homogenous fluorescence release assay. Purified recombinant enzymes were incubated with serial-diluted inhibitors at the indicated concentrations, with pre-incubation periods ranging from 0 to 3 hrs, in the standard HDAC buffer. Acetyl-Lys(Ac)-AMC substrate (at 10 μM, corresponding to the Km for both HDAC1 and HDAC3) was added after the pre-incubation period. The reaction was allowed to run for 1 hr. The trypsin peptidase developer, at final concentration of 5 mg/mL, was added after 1 hr, and the fluorescence emission was then measured using a Tecan M200 96-well plate reader.

Cell lines

HH and Hut78 CTCL cells

Preparation method

The solubility of this compound in DMSO is >18.1 mg/mL. 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

~ 10 μM; 24 ~ 72 hrs

Applications

In HH and Hut78 CTCL cells, the 24-hr RGFP966 treatment increased the acetylation of H3K9/K14, H3K27, and H4K5, but not H3K56ac. In addition, RGFP966 also inhibited the growth of CTCL cells by increasing apoptosis that was related to DNA damage and impaired S phase progression.

Animal models

C57BL/6J mice

Dosage form

3, 10 and 30 mg/kg; systemic delivery

Applications

After ORM training, RGFP966 significantly increased preference for the novel object in a dose-dependent manner. Mice treated with 10 mg/kg RGFP966 either 1 hr before or after subthreshold OLM training showed enhanced preference for the object in a novel location during the retention test, without any effect on the total exploration time of objects.

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.

RGFP966 is a selective inhibitor of HDAC3 with IC50 value of 80 nM [1].

HDAC3 (histone deacetylase 3) is an isoform of HDACs family and plays an important role in DNA transcriptional regulation. It has been shown that HDACs involves in a variety of biologic processes, like DNA repair, replication, transcription and chromatin structure. Many studies have shown that HDACs is the most highly expressed class I HDAC in the brain and involves in learning and memory process negatively [2].

RGFP966 is a potent HDAC3 inhibitor and has no inhibition on other HDACs at the concentration up to 15 μM. Using a substrate-dependent biochemical assay to investigate RGFP966 inhibition of HDACs, and results showed that it inhibited HDAC3 with IC50 value of 80 nM while had no effective inhibition on other HDACs [1]. When tested with cutaneous T cell lymphoma (CTCL) cell lines with RGFP966, the cell growth were significantly decreased by inhibiting HDAC3 which increased cell apoptosis [3].

In male C57BL/6J mice model, trained them in object recognition (ORM) and location-dependent object recognition (OLM) with a subthreshold period after administration of RGFP966 significantly increased the novel object preference and enhanced enhanced long-term OLM in a dose-dependent manner [1].

References:
[1]. Malvaez, M., et al., HDAC3-selective inhibitor enhances extinction of cocaine-seeking behavior in a persistent manner. Proc Natl Acad Sci U S A, 2013. 110(7): p. 2647-52.
[2]. Rogge, G.A., et al., HDAC3 is a negative regulator of cocaine-context-associated memory formation. J Neurosci, 2013. 33(15): p. 6623-32.
[3]. Wells, C.E., et al., Inhibition of histone deacetylase 3 causes replication stress in cutaneous T cell lymphoma. PLoS One, 2013. 8(7): p. e68915.