Screening of histone deacetylase 1 inhibitors in natural products by capillary electrophoresis
Screening of histone deacetylase 1 inhibitors in natural products by capillary electrophoresis
复制标题
毛细管电泳筛选天然产物中组蛋白脱乙酰酶1抑制剂
DOI：
10.1039/c7ay01428g
复制
发表时间：
2017-09
影响因子：
3.1
通讯作者：
Kang Jingwu
中科院分区：
文献类型：
--
作者：
Zhang Yanmei;Li Feng;Kang Jingwu
建立了用毛细管电泳（CE）联合激光诱导荧光（LIF）检测筛选天然产物中组蛋白去乙酰化酶1 （HDAC1）抑制剂的方法。该方法采用5-羧基荧光素标记肽和乙酰化赖氨酸残基作为HDAC1的底物，并建立了一个由38种纯化天然产物组成的小化学文库。生化实验采用CE分离法，即将酶促反应溶液中的去乙酰化产物与底物肽分离；因此，通过测量去乙酰化产物的峰面积，可以准确地计算出酶的活性。为了筛选抑制剂，将测试样品加入底物溶液中，然后将得到的溶液混合物与酶溶液一起在30°C下孵育短时间，进行去乙酰化反应。将反应瓶置于沸水中淬灭反应后，用含有30 mM NaCl和0.05% （m/v）六甲基溴（HDB）的100 mM Tris-H3PO4缓冲液（pH 8.0）注入CE分离。只要脱乙酰化产物的峰面积比阴性对照在没有任何抑制剂的情况下减少，就可以很容易地识别出抑制剂。为了减少底物和产物多肽在毛细管壁上的严重吸附，在毛细管壁上动态涂覆带正电荷的HDB是非常必要的。提出了一种快速、经济的HDAC1抑制剂筛选方法。
A method for the screening of histone deacetylase 1 (HDAC1) inhibitors in natural products by using capillary electrophoresis (CE) coupled with laser induced fluorescence (LIF) detection was developed. The method was developed by employing a 5-carboxyfluorescein labelled peptide with an acetylated lysine residue as the substrate of HDAC1 and a small chemical library composed of 38 purified natural products. The biochemical assay was performed by means of CE separation, i.e. the deacetylated product in the enzymatic reaction solution was separated from the substrate peptide; therefore, the enzyme activity can be accurately calculated through the measurement of the peak area of the deacetylated product. For inhibitor screening, the tested samples were spiked in the substrate solution and the resulting solution mixtures were then incubated with the enzyme solution for proceeding the deacetylation reaction for a short period of time at 30 °C. After quenching the reaction by putting the reaction vial in boiling water, the resulting solution was injected for CE separation with 100 mM Tris–H3PO4 buffer (pH 8.0) containing 30 mM NaCl and 0.05% (m/v) hexadimethrine bromide (HDB). The inhibitor could be readily identified as long as the peak area of the deacetylated product is reduced in comparison with that of the negative control in the absence of any inhibitor. Dynamically coating the capillary wall with positively charged HDB is very necessary to diminish the serious adsorption of the substrate and product peptides onto the capillary wall. A rapid, cost-effective method for HDAC1 inhibitor screening is proposed.
登录
查看更多内容
影响因子：
2.9
作者：
Jingwu Kang;D. Wistuba;V. Schurig
通讯作者：
Jingwu Kang;D. Wistuba;V. Schurig
毛细管电泳前沿分析筛选蛋白质-蛋白质相互作用的小分子抑制剂
影响因子：
7.4
作者：
Xu Mei;Liu Chao;Zhou Mi;Li Qing;Wang Renxiao;Kang Jingwu
通讯作者：
Kang Jingwu
影响因子：
46.9
作者：
Bantscheff, Marcus;Hopf, Carsten;Drewes, Gerard
通讯作者：
Drewes, Gerard
影响因子：
4.8
作者：
Chou, C. James;Herman, David;Gottesfeld, Joel M.
通讯作者：
Gottesfeld, Joel M.
电泳介导微量分析技术筛选天然提取物中的乙酰胆碱酯酶抑制剂
影响因子：
2.9
作者：
Tang, Zhong-mei;Wang, Zhao-yan;Kang, Jing-wu
通讯作者：
Kang, Jing-wu