生物活性

GSK343 is a potent, specific inhibitor of the histone H3-lysine 27 methyltransferase ENX-1 (EZH2). GSK343 inhibits ENX-1 enzymatic activity with an IC50 of 4 nM. The compound displays 60 fold selectivity for ENX-1 vs. EZH1, and 1000 fold or greater selectivity against other histone methyltransferases. The IC50 for inhibition of H3K27 methylation is < 200 nM in HCC1806 cells.

The inhibition of methyltransferases[1]

Select enzyme target data[1]

Cellular activityofGSK343[1]

体外研究

体内研究

激酶实验

Invitrobiochemical assays against histonemethyltransferases.[1]

Activity against EZH2 was assessed using 5member PRC2 complex (Flag-EZH2, EED, SUZ12, AEBP2, RbAp48). The assay protocolmay be summarized as follows: 10 mM stocks of compounds were prepared fromsolid in 100% DMSO. An 11 point serial dilution master plate was prepared in384 well format (1:3 dilution, columns 6 and 18 were equal volume DMSOcontrols) and dispensed to assay ready plates using acoustic dispensingtechnology to create a 100 nL stamp of compound and DMSO controls. The assayadditions consisted of equal volume additions of 10 nM EZH2 and the substratesolution (5 μg/mL HeLa nucleosomes and 0.25 μM [3H]-SAM) dispensedinto assay plates using a multi-drop combi dispense. Reaction plates wereincubated for 1 hr and quenched with an equal volume addition of 0.5 mg/mLPS-PEI Imaging Beads containing 0.1 mM unlabeled SAM. The plates were sealed,dark adapted for 30 minutes, and a 5 minute endpoint luminescence image wasacquired using a Viewlux imager. Plate statistics such as Z’ and signal tobackground as well as dose response curves were analyzed using ActivityBaseXE.The in vitro biochemicalactivity of EZH1 was assessed as part of a 5 member PRC2 complex using a 384well SPA assay identical to EZH2. Buffer components, reagent dispensing,compound plate preparation, quench conditions and data analysis were identicalfor EZH1 and EZH2 with final assay concentrations of 20 nM EZH1, 5 μg/mL HeLanucleosomes and 0.25 μM [3H]-SAM. Further data analysis, pIC50pivots and visualizations were enabled by TIBCO Spotfire. Compounds wereprofiled at Reaction Biology Corp. to assess inhibition in their panel ofhistone methyltransferase assays. Methyltransferase activity was assessed usingHotSpot technology, a miniaturized radioisotope-based filter binding assay.Inhibitors were dissolved in dimethyl sulfoxide (DMSO) and tested at concentrationsup to 100 uM with a final DMSO concentration of 2%. Buffer containing themethyltrasferase at the listed concentration and its preferred substrate asshown in the accompanying table was preincubated with compound for 10 min.Reactions were initiated by the addition of 1 uMS-adenosyl-L-[methyl-3H]methionine (SAM), allowed to incubate for 60 min at 30oCfollowed by transfer to P81 filter-paper and PBS wash before detection.

细胞实验

Cellculture[2]

HepG2 cells were cultured in DMEM mediumsupplemented with10% FBS, 1 mM sodium pyruvate, 1% L-glutamine, 1% Antibiotic-AntimycoticSolution, and incubated at 37°C in a humidified incubator containing 5% CO2.

Cellviability assay

Cell viability was measured with an MTTassay. Cells were plated in 96-well plates and treated with drugs. After 72 hof incubation, 0.5 mg/mL of MTT was added to each well for an additional 4 h.The blue MTT formazan precipitate was then dissolved in 200 μL of DMSO. Theabsorbance at 550 nm was measured on a multiwell plate reader.

动物实验

Xenografttumours in nude mice[3]

Six-week-old female nude BALB/c mice werehoused under specific pathogen-free conditions. SiHa cells (1x 107)were suspended in 100-lL phosphate-buffered saline and then inoculatedsubcutaneously into the posterior dorsal region of each mouse. Tumour volumewas monitored every day in two dimensions with a digital caliper and calculatedas ((length) x (width)2x 0.52). To study the effect of the EZH2 inhibitorGSK343, 5 mg/kg in 100-μL phosphate-buffered saline was injected intraperitoneally everyother day into BALB/c nude mice (n = 6) after the tumour volume reached 100 mm3.In this analysis, the negative control group (n = 6) received saline. After 40days, the mice were killed, and the subcutaneous tumours were surgicallyexcised, weighed, photographed, sectioned, and fixed in 10% formalin. The expressionlevels of E-cadherin, Ncadherin, and vimentin in the tumours were measured byrealtime reverse transcription polymerase chain reaction.

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] Verma SK, Tian X, LaFrance LV, et al. Identification of Potent, Selective, Cell-Active Inhibitors of the Histone Lysine Methyltransferase EZH2. ACS Med Chem Lett. 2012;3(12):1091-1096.
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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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