LAQ824 (Dacinostat (NVP-LAQ824)) is a novel HDAC inhibitor with IC50 of 32 nM and is an activator of the p21 promoter.

HDAC:32 nM

100 mg/kg LAQ824剂量依赖性抑制携带HCT116和人结肠癌移植瘤的裸鼠肿瘤生长,且无细胞毒性.

LAQ824诱导A549细胞中p21蛋白的剂量依赖性增加和Rb肿瘤抑制因子的低磷酸化状态的增加。LAQ824在IL-10基因启动子水平诱导染色质改变,导致增强的转录抑制因子HDAC11和PU.1的募集并抑制BALB/c鼠巨噬细胞中IL-10的产生。此外,LAQ824通过激活p21启动子,也激活编码p21细胞周期抑制剂的基因表达,最大启动子活性AC50为50%时,浓度为0.30 μM。 LAQ824抑制H1299（一种非小细胞肺癌细胞系）和HCT116（一种结肠癌细胞系）的细胞生长,IC50分别为0.15 μM和0.01 μM,LAQ824的抗增殖作用对肿瘤细胞系具有选择性,同时仅在正常成纤维细胞中诱导生长停滞。

In Vitro Histone Deacetylase Assay: HDAC enzymes are partially purified from H1299 cell lysate by ion exchange chromatography using the Q Sepharose Fast Flow column. Enzyme complexes are collected from 500 mg of total cell lysate by immunoprecipitation with cdk2 polyclonal antibody or cdk1/cdc2 monoclonal antibody. Immunoprecipitates are resuspended in kinase buffer (50 mM Hepes, pH 8, 10 mM MgCl2, 2.5 mM EDTA, 1 mM dithiothreitol, 20 mM ATP, 10 mM β-glycerophosphate, 0.1 mM NaVO4, 1 mM sodium fluoride, 50 mM ATP, 10 μCi of [γ-32P]ATP) along with 1 μg of pRb recombinant protein substrate (cdk2) or 10 mL of H1 histone mixture containing 20 μg of substrate (cdc2). Phosphorylated Rb and H1 histone are resolved by electrophoresis and quantitated using a PhosphorImager.

Cell proliferation is measured using an adaptation of published procedures (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium assay). The cells are seeded in 12-well dishes and cultured in RPMI 1640 containing 10% FBS. The cells are cultured in the presence of various concentrations of TSA (up to 1,000 ng/mL). To examine the growth inhibition by TSA, viable cell numbers are determined by trypan blue dye exclusion, counted in a Nesbauer-type hemocytometer for 0 hour, 24 hours, and 48 hours. The same amount of ethanol is added to the RPMI 1640 medium as the control experiment. All experiments are performed in duplicate and repeated 3 times The average background value (treatment with medium alone) is subtracted from each experimental well; triplicate values are averaged for each compound dilution. The following formulas are used to calculate the percentage of growth: If XT0, %Growth=(X-T0)/(GC-T0)*100. where T0 is the average value of T0 ? background, GC is the average value of untreated cells (in triplicate) ? background, and X is the average value of compound-treated cells (in triplicate)-background. The "% Growth" is plotted against compound concentration and used to calculate the IC50 using the linear regression techniques between data points to predict the concentration of compounds at 50% inhibition.(Only for Reference)