组蛋白赖氨酸甲基转移酶 DOT1L 抑制剂分子水平高通量筛选模型的建立

doi:10.12066/j.issn.1007-2861.1757

上海大学学报(自然科学版) ›› 2018, Vol. 24 ›› Issue (1): 126-133.doi: 10.12066/j.issn.1007-2861.1757

组蛋白赖氨酸甲基转移酶 DOT1L 抑制剂分子水平高通量筛选模型的建立

徐威¹, 苏明波², 周宇波²()

^1. 上海大学生命科学学院, 上海 200444
^2. 国家新药筛选中心, 上海 201203

收稿日期:2016-03-21 出版日期:2018-02-28 发布日期:2018-03-05
通讯作者: 周宇波 E-mail:ybzhou@simm.ac.cn
基金资助:
国家科技部重大专项资助项目(2014ZX09507-002-004)

Establishment of molecular-based high-throughout screening model for histone lysine methyltransferase DOT1L inhibitors

XU Wei¹, SU Mingbo², ZHOU Yubo²()

^1. School of Life Sciences, Shanghai University, Shanghai 200444, China
^2. The National Center for Drug Screening, Shanghai 201203, China

Received:2016-03-21 Online:2018-02-28 Published:2018-03-05
Contact: ZHOU Yubo E-mail:ybzhou@simm.ac.cn

摘要/Abstract

摘要：

类端粒沉默干扰体 1(disruptor of telomeric silencing 1-like, DOT1L) 是一种能够催化组蛋白 H3 第 79 位赖氨酸(H3K79)发生甲基化修饰的表观遗传调控酶, 是第一个被发现不含有 SET 结构域的赖氨酸甲基转移酶. H3K79 位点的甲基化程度能够影响和调控某些特定基因的表达, 与急性髓性白血病(acute myelocytic leukemia, AML)的发生与发展密切相关. 为建立一种分子水平 DOT1L 小分子抑制剂的高通量筛选模型, 以 EPZ-5676 为阳性化合物, 探讨最佳反应酶浓度、底物浓度及反应时间等, 并对优化后的反应体系进行检测和确认. 通过对多种参数优化使得高通量筛选体系的 $Z'$ 因子达到 0.54, 表明已成功建立了 DOT1L 小分子抑制剂高通量筛选模型.

关键词: 类端粒沉默干扰体 1(disruptor of telomeric silencing 1-like, DOT1L), 组蛋白赖氨酸甲基转移酶, 小分子抑制剂, 高通量筛选

Abstract:

DOT1L is an epigenetic enzyme that can catalyze methyltransfer activity at histone 3 lysine 79 (H3K79). It is also found to be the first lysine methyltransferase without a SET domain. Research results show that methylation level of H3K79 can influence the expression of some genes, closely related to the development of acute myelocytic leukemia (AML). This work establishes a molecular-based high-throughout screening model for discovering novel DOT1L inhibitors. EPZ-5676 was used as positive control to optimize activity assay and ensure appropriate concentration of enzyme and substrate, and reaction time, etc. The assay was optimized, and the $Z'$-factor was about 0.54, indicating that a molecular-based high-throughout screening model for DOT1L inhibitors was successfully established.

Key words: disruptor of telomeric silencing 1-like (DOT1L), histone lysine methyltransferase, small molecule inhibitor, high-throughout screening (HTS)

中图分类号:

R914.4

徐威, 苏明波, 周宇波. 组蛋白赖氨酸甲基转移酶 DOT1L 抑制剂分子水平高通量筛选模型的建立[J]. 上海大学学报(自然科学版), 2018, 24(1): 126-133.

XU Wei, SU Mingbo, ZHOU Yubo. Establishment of molecular-based high-throughout screening model for histone lysine methyltransferase DOT1L inhibitors[J]. Journal of Shanghai University（Natural Science Edition）, 2018, 24(1): 126-133.

图/表 8

Table 1

图1

图2

图3

图4

图5

图6

图7

参考文献 13

[1]	Bannister A J, Kouzarides T . Regulation of chromatin by histone modifications[J]. Cell Research, 2011,21(3):381-395. doi: 10.1038/cr.2011.22
[2]	Portela A, Esteller M . Epigenetic modifications and human disease[J]. Nat Biotech, 2010,28(10):1057-1068. doi: 10.1038/nbt.1685
[3]	Wong M, Polly P, Liu T . The histone methyltransferase DOT1L: regulatory functions and a cancer therapy target[J]. American Journal of Cancer Research, 2015,5(9):2823-2837. pmid: 26609488
[4]	Min J, Feng Q, Li Z , et al. Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase[J]. Cell, 2003,112(5):711-723. doi: 10.1016/s0092-8674(03)00114-4 pmid: 12628190
[5]	Wen L, Fu L, Guo X , et al. Histone methyltransferase DOT1L plays a role in postembryonic development in $Xenopus tropicalis$[J]. FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology, 2015,29(2):385-393. doi: 10.1096/fsb2.v29.2
[6]	Stein E M, Tallman M S . Mixed lineage rearranged leukemia: pathogenesis and targeting DOT1L[J]. Current Opinion in Hematology, 2015,22(2):92-96. doi: 10.1097/MOH.0000000000000123 pmid: 25635757
[7]	Liu W, Deng L, Song Y , et al. DOT1L inhibition sensitizes MLL-rearranged AML to chemotherapy[J]. PLoS ONE, 2014,9(5):e98270. doi: 10.1371/journal.pone.0098270 pmid: 24858818
[8]	Chen C W, Armstrong S A . Targeting DOT1L and HOX gene expression in MLL-rearranged leukemia and beyond[J]. Experimental Hematology, 2015,43(8):673-684. doi: 10.1016/j.exphem.2015.05.012 pmid: 26118503
[9]	朱晓华, 冯玉田, 章玉鉴 . 基于组件技术的虚拟仪器开发方法的研究[J]. 上海大学学报(自然科学版), 1999,5(4):357-361.
[10]	Daigle S R, Olhava E J, Therkelsen C A , et al. Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor[J]. Cancer Cell, 2011,20(1):53-65. doi: 10.1016/j.ccr.2011.06.009
[11]	Daigle S R, Olhava E J, Therkelsen C A , et al. Potent inhibition of DOT1L as treatment of MLL-fusion leukemia[J]. Blood, 2013,122(6):1017-1025. doi: 10.1182/blood-2013-04-497644
[12]	Yu W, Chory E J, Wernimont A K , et al. Catalytic site remodelling of the DOT1L methyltransferase by selective inhibitors[J]. Nature Communications, 2012,3:1288. doi: 10.1038/ncomms2304 pmid: 23250418
[13]	Peters C D, Jespersen B, Norregaard R . AlphaLISA versus ELISA-based detection of interleukin 18 in healthy subjects and patients with end-stage renal disease[J]. Scandinavian Journal of Clinical and Laboratory Investigation, 2012,72(8):583-592. doi: 10.3109/00365513.2012.713175 pmid: 22935048

组蛋白赖氨酸甲基转移酶 DOT1L 抑制剂分子水平高通量筛选模型的建立

Establishment of molecular-based high-throughout screening model for histone lysine methyltransferase DOT1L inhibitors

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 13

相关文章 1

编辑推荐

Metrics

本文评价