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    LUMINESCENCE-BASED METHODS AND PROBES FOR MEASURING CYTOCHROME P450 ACTIVITY
    9.
    发明申请
    LUMINESCENCE-BASED METHODS AND PROBES FOR MEASURING CYTOCHROME P450 ACTIVITY 有权
    用于测量细胞色素P450活性的基于荧光的方法和探针

    公开(公告)号：US20110003316A1

    公开(公告)日：2011-01-06

    申请号：US12754164

    申请日：2010-04-05

    申请人： James J. Cali ,  Dieter Klaubert ,  William Daily ,  Samuel Kin Sang Ho ,  Susan Frackman ,  Erika Hawkins ,  Keith V. Wood

    发明人： James J. Cali ,  Dieter Klaubert ,  William Daily ,  Samuel Kin Sang Ho ,  Susan Frackman ,  Erika Hawkins ,  Keith V. Wood

    IPC分类号： G01N33/53 ,  C12Q1/66

    CPC分类号： G01N33/502 ,  A61K49/0008 ,  C07D417/04 ,  C12Q1/66 ,  G01N33/5088 ,  G01N33/582 ,  G01N2333/90245 ,  G01N2333/90251 ,  G01N2500/10

    摘要： The present invention provides methods, compositions, substrates, and kits useful for analyzing the metabolic activity in cells, tissue, and animals and for screening test compounds for their effect on cytochrome P450 activity. In particular, a one-step and two-step methods using luminogenic molecules, e.g. luciferin or coelenterazines, that are cytochrome P450 substrates and that are also bioluminescent enzyme, e.g., luciferase, pro-substrates are provided. Upon addition of the luciferin derivative or other luminogenic molecule into a P450 reaction, the P450 enzyme metabolizes the molecule into a bioluminescent enzyme substrate, e.g., luciferin and/or luciferin derivative metabolite, in a P450 reaction. The resulting metabolite(s) serves as a substrate of the bioluminescent enzyme, e.g., luciferase, in a second light-generating reaction. Luminescent cytochrome P450 assays with low background signals and high sensitivity are disclosed and isoform selectivity is demonstrated. The present invention also provides an improved method for performing luciferase reactions which employs added pyrophosphatase to remove inorganic pyrophosphate, a luciferase inhibitor which may be present in the reaction mixture as a contaminant or may be generated during the reaction. The present method further provides a method for stabilizing and prolonging the luminescent signal in a luciferase-based assay using luciferase stabilizing agents such as reversible luciferase inhibitors.

    摘要翻译： 本发明提供了可用于分析细胞，组织和动物中的代谢活性以及筛选测试化合物对细胞色素P450活性的影响的方法，组合物，底物和试剂盒。 特别地，使用发光分子的一步和两步法。 荧光素或coelenterazines，其是细胞色素P450底物并且也是生物发光酶，例如萤光素酶，原底物。 在P450反应中加入荧光素衍生物或其他发光分子时，P450酶在P450反应中将分子代谢为生物发光酶底物，例如荧光素和/或荧光素衍生物代谢产物。 所产生的代谢物在第二次发光反应中用作生物发光酶，例如荧光素酶的底物。 公开了具有低背景信号和高灵敏度的发光细胞色素P450测定，证明了同工型选择性。 本发明还提供一种用于进行荧光素酶反应的改进方法，其使用添加的焦磷酸酶来除去无机焦磷酸盐，荧光素酶抑制剂，其可作为污染物存在于反应混合物中，或可能在反应期间产生。 本发明方法还提供了一种使用荧光素酶稳定剂如可逆荧光素酶抑制剂在基于荧光素酶的测定中稳定和延长发光信号的方法。