生物活性

BIX01294, a G9a histone methyltransferase (HMT)-specific inhibitor, inducesapoptosis and inhibits the proliferation, migration, and invasion of cancercells. BIX01294 inhibits G9a. BIX01294 is an inhibitor of HDAC. The histone methyltransferase (HMTase) G9a can mono- or dimethylate lysine 9 on histone 3 (H3), contributing to early embryogenesis, genomic imprinting, and lymphocyte development. BIX01294 is a selective inhibitor of G9a HMTase (IC50 = 1.7 μM). It less effectively inhibits the HMTase G9a-like protein (GLP; IC50 = 38 μM) and has no effect on other known HMTases. BIX01294 has been used in combination with the calcium channel activator BayK8644 to facilitate the generation of induced pluripotent stem cells from somatic cells in vitro. Modulates H3K9me2 levels in mammalian cells and potentiates induction of pluripotent stem cells from somatic cells in vitro.

HMTaseactivities of BIX01294[1]

体外研究

体内研究

激酶实验

HMTase Assays on Microtiter Plates[1]

Dissociation Enhanced LanthanideFluoro-Immuno Assays (DELFIA) were performed in white, opaque 384-well platescoated with Neutravidin. Test compounds were diluted to 12μg/ml in 50mM Tris-HCl pH 8.5 containing 4% DMSO and 10μl was dispensed into the wells. Blank and control wells receivedonly compound buffer. GST-G9a at 10μg/ml and SAM at 40μM were diluted in 50mMTris HCl pH 8.5/10mM DTT and added in a volume of 20μL. Blank wells received Tris/DTTbuffer only. The reactions were initiated by the addition of 800nM H3 (1-20)-cysbiotinsubstrate in 50mM Tris pH 8.5 in a volume of 10μl, and incubated at room temperature for 60 minutes. The plates werewashed 3 times with 100μl of Wash Buffer (50mMTris pH 7.4, 150mM NaCl, 0.05% Tween 20, 0.2% BSA). Next, 50μl of Fluoroimmunoassay (FI) Buffer (50mM Tris HCl pH 7.8, 150mMNaCl, 0.05% Tween 40, 25μM DTPA, 0.2% BSA, 0.05%BGG) containing 5ng α-2X-di-meth H3-K9 and5ng goat anti-rabbit Eu chelate was added to all wells of the plate, and theplate was incubated for an additional hour at room temperature. The plates werewashed 3 times with 100μl of Wash Buffer, and 50μl of Enhancement Solution was added to each well. Time resolvedfluorescence was measured after 45 minutes on a Viewlux Microplate Imagerimaging for 15 seconds with a 354μswindow, 400μs delay, excitation at360 nm, and emission at 618 nm.

细胞实验

Cell and culture conditions[2]

Human glioblastoma cell line U251 wasmaintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum (FBS) and 5% antibiotic‑antimycotic (Gibco). Cells were cultured at 37˚C in a humidifiedatmosphere of 5% CO₂.

Drugtreatment

Stock solutions of 10mM BIX01294 (Bix) weredissolved in dimethyl sulfoxide (DMSO) and diluted in culture medium to 1, 2, 5and 10μM for cell treatment. For a single treatment of Bix (SBT), the cellswere incubated at 37˚C overnight, and subsequently treated with 1, 2, 5 and 10μMfor 1 day (24 h). For the sequential treatment of Bix (SeBT), the cells wereincubated at 37 ˚C overnight, and treated with 1μM Bix. Bix and medium wasreplaced every 2 days for 2 weeks.

Cellproliferation assay

Cell proliferation was assessed using themethyl thiazolyl tetrazolium (MTT) colorimetric assay. U251 cells (2x10⁵cells/well)were seeded in 6‑well plates and incubated at 37˚C overnight. Following24h of incubation, the medium was removed and replaced with the experimentalmedium. Cells were treated with 1, 2, 5 and 10μM of Bix for SBT or 1μM of Bixfor SeBT. Subsequently, cells were washed twice with phosphate‑buffered saline (PBS), and 5mg/ml MTT diluted in PBS was added toeach well for a total of 4 h. Following removal of the MTT solution,solubilization solution (DMSO/ethanol, 1:1 ratio) was added to each well todissolve the formazan crystals. The absorbance at 570nm was measured using aParadigm™ Detection Platform  andanalyzed using Multimode Analysis Software version 3.3.0.9.

动物实验

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Kubicek S, O'Sullivan RJ, August EM, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase. Mol Cell. 2007;25(3):473-481.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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