HDAC activity

HDAC activity was measured using a continuous trypsin-coupled assay. For inhibitor characterization, measurements were done in a reaction volume of 100 μL using 96-well assay plates. For each isozyme, the HDAC protein in reaction buffer [50 mM HEPES, 100 mM KCl, 0.001% Tween 20, 5% DMSO (pH 7.4), supplemented with bovine serum albumin at concentrations of 0% (HDAC1), 0.01% (HDAC2, 3, 8, and 10), or 0.05% (HDAC6)] was mixed with PCI-24781 at various concentrations and allowed to incubate for 15 mins. Trypsin was added to a final concentration of 50 nM, and acetyl-Gly-Ala-(N-acetyl-Lys)-AMC was added to a final concentration of 25 μM (HDAC1, 3, and 6), 50 μM (HDAC2 and 10), or 100 μM (HDAC8) to initiate the reaction. Negative control reactions were done in the absence of PCI-24781 in replicates of eight. Reactions were monitored in a fluorescence plate reader. After a 30-min lag time, the fluorescence was measured over a 30-min time frame using an excitation wavelength of 355 nm and a detection wavelength of 460 nm. The increase in fluorescence with time was used as the measure of the reaction rate. Inhibition constants Ki(app) were obtained using the program BatchKi.

Cell lines

HCT116, DLD-1, HCT-15, MCF-7, BT-549, NCI-H226, CWR-22RV1, NCI-PC3, SKOV-3, OVCAR-3 and HUVEC cell lines

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.

Reaction Conditions

~ 10 μM; 48, 72, 96, or 120 hrs

Applications

PCI-24781 exhibited potent antitumor activity against a variety of tumor cell lines with the GI50% values ranging from 0.15 μM to 3.09 μM. In addition, PCI-24781 also showed an antiproliferative effect on HUVEC with the GI50% value of 0.43 μM.

Animal models

Female BALB/c nu/nu mice implanted s.c. with HCT116 and DLD-1 tumor cells

Dosage form

200 mg/kg; i.v.; q.d., every other day

Applications

Administration of PCI-24781 (200 mg/kg; i.v.; q.d., every other day) significantly inhibited the growth of HCT116 and DLD-1 xenografts in mice by 69% and 59%, respectively.

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.

CRA-024781 is a novel HDAC inhibitor targeting HDAC1, HDAC2, HADC3, HADC6, HADC8, HADC10 with IC50 value of 7 nM(Ki), 19 nM(Ki), 8.2 nM(Ki), 17 nM(Ki), 280 nM(Ki), 24 nM(Ki), respectively [1].

Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups (O=C-CH3) from an ε-N-acetyl lysine amino acid on a histone, allowing the histones to wrap the DNA more tightly.

CRA-024781 exhibits potent antitumor activity against ten human tumor cell lines with GI50 ranging from 0.15 μM to 3.09 μM. CRA-024781 also has an antiproliferative effect on HUVEC endothelial cells with GI50 of 0.43 μM. CRA-024781 treatment causes dose-dependent accumulation of both acetylated histones and acetylated tubulin in HCT116 or DLD-1 cells suggesting that HDAC enzymes are inhibited in these cells. CRA-024781 also induces the expression of p21 (a protein playing a role in the antitumor effect) and leads to PARP cleavage and accumulation of the γH2AX, which indicate apoptosis [1].

Treatment with CRA-024781 at 200 mg/kg once daily every other day significantly inhibits the growth of HCT116 and DLD-1 xenografts in mice by 69% and 59%, respectively. In the HCT116 model, treatment with CRA-024781 at 20 mg/kg, 40 mg/kg, 80 mg/kg, or 160 mg/kg (q.d. × 4 per week) causes inhibition of tumor growth by 48%, 57%, 82.2%, or 80.0%, respectively [1].

Reference:
[1].  Buggy JJ, Cao ZA, Bass KE, et al. CRA-024781: a novel synthetic inhibitor of histone deacetylase enzymes with antitumor activity in vitro and in vivo. Mol Cancer Ther, 2006, 5(5): 1309-1317.
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