生物活性

SGC-0946 is a highly selective small-moleculeDOT1L inhibitor, reduces the expression of the DOT1L and N-Myc target gene E2F2against MYCN-amplified neuroblastoma. SGC-0946 is an inhibitor of DOT1L1. SGC 0946 is over 100-fold selective for other histone methyltransferases/HMTs. SGC 0946 potently reduces H3K79 dimethylation (IC50= 2.6 nM) in A431 cells, and 8.8 nM in MCF10A cells, which potently and selectively kills cells containing an MLL translocation. SGC 0946 is much more potent than its close analog EPZ004777.

DOT1Linhibitory potency of SGC0946[1]

SGC0946reduces H3K79 methylation levels in MCF10A cells with an IC50 of 8.8 ± 1.6 nM[1]

体外研究

体内研究

激酶实验

DOT1L methyltransferase assay[1]

The reported assay condition was used with minormodification. Nucleosomes purified from chicken blood cells were used as substrate.Sixty nanomolar nucleosome solutions was added into 40 ml assay buffer (20mMTris–HCl, 2mM DTT, 10mM MgCl2 and 0.01% Triton X-100), which contained 0.25nMrecombinant DOT1L (1–420) with inhibitors of different concentrations (ordimethylsulphoxide (DMSO) as control). A similar 40 ml mixture without DOT1Lwas prepared as background. The mixture was incubated at room temperature for30 min before 0.75 mM 3H-SAM was added to start the reaction. The reactionmixture was incubated at room temperature for 2 h and quenched by the additionof 160 ml 10% trichloroacetic acid. The mixture was transferred into glassfibre filter plates and washed twice with 10% trichloroacetic acid. An ethanolwash (100 ml) was followed by drying. One hundred microlitre Microscint Zerowas finally added into each well of the filter plates and centrifuged to flowthrough filters. Tritium incorporation was detected on a TopCount.

细胞实验

Cell culture[2]

Neuroblastoma BE(2)-C, NBL-S, SK-N-FI, andHEK-293 cells were cultured in DMEM supplemented with 10% FCS. Kelly cells weregrown in RPMI1640 supplemented with 10% FCS.

Proteincoimmunoprecipitation assays

HEK293 human embryonic cells weretransiently cotransfected with an N-Myc-expression pcDNA3.1 construct, togetherwith an empty vector or Flag-DOT1L-expression pcDNA3.1 construct for 48 hours.Protein was extracted from the cells and coimmunoprecipitation was carried outusing an anti-FLAG antibody or a mouse IgG as a negative control, followed by immunoblotanalysis.

GSTpull-down assays

Different N-Myc protein fragments werecloned into the pGEX-2T construct, in frame with N-terminal GST. The constructswere transformed into BL-21 E.Coli and IPTG was used for induction of T7-driventranscription. After purification, equal amount of GSTN- Myc protein fragmentswere immobilized onto glutathioneagarose beads. HEK-293T cells were transfectedwith Flag-DOT1L expression construct, and nuclear protein from the cells wasincubated with equal amount of different GST-N-Myc protein fragmentsimmobilized onto glutathione agarose beads. Pulleddown complexes were analyzedby immunoblot with a monoclonal anti-Flag antibody, and Ponceau stainingdetected by ChemiDoc MP was used as loading controls.

Chromatinimmunoprecipitation assays

Chromatin immunoprecipitation (ChIP) assayswere performed with mouse anti-N-Myc antibody, rabbit anti-H3K79me2 antibody(Abcam), rabbit and mouse control IgG, followed by PCR with primers designed tocover the core promoter regions of the DOT1L, ODC1, and E2F2 genes containingMyc-responsive element E-Boxes or remote negative control regions. Foldenrichment of the DOT1L, E2F2, and ODC1 gene core promoter regions by theanti-N-Myc or anti-H3K79me2 antibody was calculated by dividing cycle thresholdof PCR products from the DOT1L, E2F2, and ODC1 gene core promoter regions bycycle threshold of PCR products from the negative control region, relative toinput.

Luciferasereporter assays

Modulation of DOT1L promoter activity byN-Myc was analyzed by luciferase assays. The DOT1L gene core promotercontaining the E-Box (389 bp to þ50 bp relative to transcription start site)was cloned into the pLightSwitch_Prom construct. BE(2)-C cells werecotransfected with control siRNA or N-Myc siRNA-1, together with Go ClonePromoter Promoter Control construct plus the pLightSwitch_Prom construct expressingempty vector or the DOT1L core promoter. Luciferase activities were measuredwith a LightSwitch Dual Assay System Kit, and normalized according to Go Clone PromoterControl construct according to the manufacturer's instructions.

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] Yu W, Chory EJ, Wernimont AK, et al. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors. Nat Commun. 2012;3:1288.
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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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