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    Method for detecting compounds modulating dimers of VFT domain membrane proteins
    3.
    发明授权
    Method for detecting compounds modulating dimers of VFT domain membrane proteins 有权
    检测调节VFT结构域膜蛋白二聚体的化合物的方法

    公开(公告)号：US08697380B2

    公开(公告)日：2014-04-15

    申请号：US13266222

    申请日：2010-04-29

    申请人： Etienne Doumazane ,  Jurriaan Zwier ,  Eric Trinquet ,  Jean-Phillippe Pin

    发明人： Etienne Doumazane ,  Jurriaan Zwier ,  Eric Trinquet ,  Jean-Phillippe Pin

    IPC分类号： C12Q1/02 ,  C12Q1/34 ,  C12Q1/48

    CPC分类号： G01N33/542 ,  G01N33/9426 ,  G01N2333/726 ,  G01N2500/04

    摘要： The invention relates to a method for selecting compounds having a modulating effect on the activation state of a dimer of VFT-domain proteins expressed in cell membranes present in a measuring medium, said dimer consisting of a first protein and of a second protein, said proteins being identical or different, wherein this method comprises the following steps: (a) labeling the first and second proteins in the N-terminal portion of their VFT domains with the members of a pair of FRET partners, the Förster radius (R0) of said pair being between 20 and 55 Å; (b) measuring the FRET signal in the absence and in the presence of the test compound within a predetermined time window; (c) selecting the test compound as a modulating compound if a difference in FRET signal in the absence and in the presence of test compound is measured in step (b). The invention can be used in the search for new medicaments and new taste modulators.

    摘要翻译： 本发明涉及一种选择对存在于测量介质中的细胞膜中表达的VFT结构域蛋白的二聚体的活化状态具有调节作用的化合物的方法，所述二聚体由第一种蛋白质和第二种蛋白质组成，所述蛋白质 相同或不同，其中该方法包括以下步骤：（a）用一对FRET伴侣的成员将其VFT结构域的N末端部分中的第一和第二蛋白质标记，所述第一和第二蛋白质的所述F 对在20和55之间; （b）在预定时间窗内测试化合物不存在和存在时测量FRET信号; （c）如果在步骤（b）中测定了在不存在和存在测试化合物的情况下FRET信号的差异，则选择测试化合物作为调节化合物。 本发明可用于寻找新的药物和新的调味剂。

    Method for detecting membrane protein internalization
    5.
    发明授权
    Method for detecting membrane protein internalization 有权
    膜蛋白内化检测方法

    公开(公告)号：US08999653B2

    公开(公告)日：2015-04-07

    申请号：US13056738

    申请日：2009-07-30

    申请人： Jurriaan Zwier ,  Robert Poole ,  Herve Ansanay ,  Michel Fink ,  Eric Trinquet

    发明人： Jurriaan Zwier ,  Robert Poole ,  Herve Ansanay ,  Michel Fink ,  Eric Trinquet

    IPC分类号： G01N33/53 ,  C12Q1/00 ,  G01N21/76 ,  G01N33/542 ,  G01N33/50 ,  G01N33/58

    CPC分类号： G01N33/542 ,  G01N33/5035 ,  G01N33/58 ,  G01N2458/40 ,  Y10T436/13

    摘要： The instant invention provides for methods for detecting the internalization of a transmembrane protein of interest expressed at the surface of a cell. More specifically, the methods involve (a) labelling the protein of interest with a fluorescent metal complex, the lifetime of which is greater than 0.1 ms, (b) adding to the reaction medium a composition capable of causing the internalization of the protein of interest, (c) adding to the reaction medium (1) a modulating agent which is a fluorescent FRET acceptor compound compatible with the fluorescent metal complex, the final concentration of which in the reaction medium is greater than 10−7M; or (2) a reducing agent, the redox potential of which is less than +0.1 V and preferably between 0.25 and 0.75 V; or (3) an agent which binds specifically, by non-covalent bonding, with the fluorescent metal complex; (d) adding a metal ion which competes with the rare earth so as to form a non-fluorescent metal complex; (d) measuring the luminescence emitted by the reaction medium at the emission wavelength of the fluorescent metal complex and/or at the emission wavelength of the modulating compound when the compound is a fluorescent acceptor compound; and (e) comparing the signal measured in step d) with a reference signal measured on cells having been subjected only to steps a) and c).

    摘要翻译： 本发明提供了用于检测在细胞表面表达的感兴趣的跨膜蛋白的内在化的方法。 更具体地说，所述方法包括（a）用荧光金属络合物标记感兴趣的蛋白质，其寿命大于0.1ms，（b）向反应介质中加入能够引起感兴趣的蛋白质内化的组合物 ，（c）向反应介质（1）中加入调节剂，其是与荧光金属络合物相容的荧光FRET受体化合物，其最终浓度在反应介质中的浓度大于10-7M; 或（2）还原剂，其氧化还原电位小于+ 0.1V，优选0.25-0.75V; 或（3）通过非共价键合与荧光金属络合物特异性结合的试剂; （d）加入与稀土竞争的金属离子以形成非荧光金属络合物; （d）当化合物是荧光受体化合物时，测量在荧光金属络合物的发射波长处和/或调节化合物的发射波长处的反应介质发射的发光; 和（e）将步骤d）中测量的信号与在仅经受步骤a）和c）的细胞上测量的参考信号进行比较。

    METHOD FOR DETECTING MEMBRANE PROTEIN INTERNALIZATION
    6.
    发明申请
    METHOD FOR DETECTING MEMBRANE PROTEIN INTERNALIZATION 有权
    检测膜蛋白内含物的方法

    公开(公告)号：US20120009599A1

    公开(公告)日：2012-01-12

    申请号：US13056738

    申请日：2009-07-30

    申请人： Jurriaan Zwier ,  Robert Poole ,  Herve Ansanay ,  Michel Fink ,  Eric Trinquet

    发明人： Jurriaan Zwier ,  Robert Poole ,  Herve Ansanay ,  Michel Fink ,  Eric Trinquet

    IPC分类号： G01N21/64

    CPC分类号： G01N33/542 ,  G01N33/5035 ,  G01N33/58 ,  G01N2458/40 ,  Y10T436/13

    摘要： A method for detecting the internalization of a transmembrane protein of interest expressed at the surface of a cell, comprising the following steps: a) labelling the protein of interest with a fluorescent metal complex, the lifetime of which is greater than 0.1 ms; b) adding to the reaction medium a composition capable of causing the internalization of the protein of interest; c) adding to the reaction medium a modulating agent selected from: a. a fluorescent or nonfluorescent FRET acceptor compound compatible with said fluorescent metal complex, the final concentration of which in the reaction medium is greater than 10−7M; b. a reducing agent, the redox potential of which is less than +0.1 V and preferably between 0.25 and 0.75 V; c. an agent which binds specifically, by noncovalent bonding, with the fluorescent metal complex; d. a metal ion which competes with the rare earth so as to form a nonfluorescent metal complex; d) measuring the luminescence emitted by the reaction medium at the emission wavelength of the fluorescent metal complex and/or at the emission wavelength of the modulating compound when said compound is a fluorescent acceptor compound; e) comparing the signal measured in step d) with a reference signal measured on cells having been subjected only to steps a) and c).

    摘要翻译： 一种用于检测在细胞表面表达的目标跨膜蛋白的内在化的方法，包括以下步骤：a）用荧光金属络合物标记感兴趣的蛋白质，其寿命大于0.1ms; b）向反应介质中加入能够引起目的蛋白质内化的组合物; c）向反应介质中加入选自以下的调节剂：a。 与所述荧光金属络合物相容的荧光或非荧光FRET受体化合物，其最终浓度在反应介质中大于10-7M; b。 还原剂，其氧化还原电位小于+0.1V，优选0.25至0.75V; C。 通过非共价键合与荧光金属络合物特异性结合的试剂; d。 与稀土竞争以形成非荧光金属络合物的金属离子; d）当所述化合物是荧光受体化合物时，测量在荧光金属络合物的发射波长处和/或调节化合物的发射波长处的反应介质发射的发光; e）将步骤d）中测量的信号与在仅经受步骤a）和c）的细胞上测量的参考信号进行比较。

    METHOD FOR DETECTING COMPOUNDS MODULATING DIMERS OF VFT DOMAIN MEMBRANE PROTEINS
    8.
    发明申请
    METHOD FOR DETECTING COMPOUNDS MODULATING DIMERS OF VFT DOMAIN MEMBRANE PROTEINS 有权
    检测化合物调节VFT域膜蛋白二聚体的方法

    公开(公告)号：US20120115176A1

    公开(公告)日：2012-05-10

    申请号：US13266222

    申请日：2010-04-29

    申请人： Etienne Doumazane ,  Jurriaan Zwier ,  Eric Trinquet ,  Jean-Phillippe Pin

    发明人： Etienne Doumazane ,  Jurriaan Zwier ,  Eric Trinquet ,  Jean-Phillippe Pin

    IPC分类号： C12Q1/02 ,  C12Q1/34 ,  C12Q1/48

    CPC分类号： G01N33/542 ,  G01N33/9426 ,  G01N2333/726 ,  G01N2500/04

    摘要： The invention relates to a method for selecting compounds having a modulating effect on the activation state of a dimer of VFT-domain proteins expressed in cell membranes present in a measuring medium, said dimer consisting of a first protein and of a second protein, said proteins being identical or different, wherein this method comprises the following steps: (a) labeling the first and second proteins in the N-terminal portion of their VFT domains with the members of a pair of FRET partners, the Förster radius (R0) of said pair being between 20 and 55 Å; (b) measuring the FRET signal in the absence and in the presence of the test compound within a predetermined time window; (c) selecting the test compound as a modulating compound if a difference in FRET signal in the absence and in the presence of test compound is measured in step (b). The invention can be used in the search for new medicaments and new taste modulators.

    摘要翻译： 本发明涉及一种选择对存在于测量介质中的细胞膜中表达的VFT结构域蛋白的二聚体的活化状态具有调节作用的化合物的方法，所述二聚体由第一种蛋白质和第二种蛋白质组成，所述蛋白质 相同或不同，其中该方法包括以下步骤：（a）用一对FRET伴侣的成员将其VFT结构域的N末端部分中的第一和第二蛋白质标记，所述第一和第二蛋白质的所述F 对在20和55之间; （b）在预定时间窗内测试化合物不存在和存在时测量FRET信号; （c）如果在步骤（b）中测定了在不存在和存在测试化合物的情况下FRET信号的差异，则选择测试化合物作为调节化合物。 本发明可用于寻找新的药物和新的调味剂。