色谱 ›› 2013, Vol. 31 ›› Issue (7): 646-655.DOI: 10.3724/SP.J.1123.2013.04032

• 特别策划:庆祝欧庆瑜先生八十华诞专栏 • 上一篇    下一篇

细管电泳-激光诱导荧光检测用于多个胞内蛋白激酶的抑制剂筛选和选择性评价

张倩倩, 张雪佩, 张含智, 康经武   

  1. 中国科学院上海有机化学研究所, 上海 200032
  • 收稿日期:2013-04-18 出版日期:2013-07-28 发布日期:2013-07-17
  • 通讯作者: 康经武
  • 基金资助:

    National Natural Science Foundations of China (90713021, 20975109, 20675086); National Key Laboratory of the Organic Biochemistry Opening Foundation.

Inhibitor screening and selectivity assessment against multiple cellular protein kinases by capillary electrophoresis with laser-induced fluorescence detection

ZHANG Qianqian, ZHANG Xuepei, ZHANG Hanzhi, KANG Jingwu   

  1. Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
  • Received:2013-04-18 Online:2013-07-28 Published:2013-07-17

摘要:

发展了一种基于毛细管电泳(CE)-激光诱导荧光(LIF)检测的多个细胞内源激酶的抑制剂平行筛选及选择性评价方法。CE高效的分离能力和LIF检测器的高选择性,使得同时测试多个胞内激酶的活性成为可能。共4种细胞系、3种特异性蛋白激酶底物肽、2种选择性蛋白激酶抑制剂和1种非选择性蛋白激酶抑制剂用于方法的建立。特异性底物肽与细胞裂解液混合后孵育,被其相应的激酶选择性地磷酸化,利用CE-LIF分离检测磷酸化产物和底物肽。同时测定一个抑制剂对几种蛋白激酶的抑制活性,用于评价抑制剂的选择性。与传统的单靶标筛选模式相比,这种基于细胞裂解液的多靶标筛选方法能提供更多的信息,更加高效,且细胞裂解液作为一种廉价的激酶来源大大降低了筛选成本。

关键词: 蛋白激酶抑制剂筛选, 激光诱导荧光检测, 毛细管电泳, 选择性评价

Abstract:

A method that can be used for screening protein kinase inhibitors (PKIs) and simultaneously assessing their selectivity is described. The method is based on simultaneously assaying multiple cellular protein kinases by performing capillary electrophoresis (CE) separation and measuring the peak areas of the phosphorylated substrate peptides. The powerful separation capability of CE combined with the highly sensitive and selective laser-induced fluorescence (LIF) detector enables the direct screening of PKIs against cell lysates, which are used as an inexpensive source of enzymes. Four cell lines, three specific substrate peptides labeled with 5-carboxyfluorescein (5-FAM), two relative specific PKIs (TBB and H-89) and one non-specific PKI (staurosporine) were utilized to prove the methodology. With this method, the inhibitory activity of the tested compounds against multiple protein kinases was identified in parallel by comparing the peak areas of the phosphorylated substrates with those obtained in the absence of any inhibitors. The reduced peak area of the phosphorylated substrate definitively represents a positive screening result. Simultaneously, assaying the inhibition of one inhibitor against mutiple cellular protein kinases enables the assessment of its selectivity. Compared to the conventional, single-target screening format, the cell lysate-based multi-target method is more informative, more straightforward and more cost effective.

Key words: capillary electrophoresis (CE), laser-induced fluorescence detection, protein kinase inhibitor screening, selectivity assessment

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