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1. 发明授权

US06893827B1 Receptor function assay for G-protein coupled receptors and orphan receptors by reporter enzyme mutant complementation 失效
标题翻译：通过报告酶突变体互补对G蛋白偶联受体和孤儿受体的受体功能测定
公开(公告)号：US06893827B1
公开(公告)日：2005-05-17
申请号：US09654499
申请日：2000-09-01
申请人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-Jia Chang
发明人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-Jia Chang
IPC分类号： C12N15/09 , C12N1/15 , C12N1/19 , C12N1/21 , C12N5/10 , C12P21/04 , C12Q1/02 , C12Q1/34 , G01N33/566 , C07K14/705 , C07K19/00 , C12N15/62 , G01N33/567
CPC分类号： G01N33/566 , G01N2333/726 , G01N2500/10
摘要： Methods for detecting G-protein coupled receptor (GPCR) activity; methods of assaying GPCR activity; and methods of screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described.
摘要翻译：检测G蛋白偶联受体（GPCR）活性的方法; 测定GPCR活性的方法; 并描述了筛选GPCR配体，G蛋白偶联受体激酶（GRK）活性和与GPCR调节过程的组分相互作用的化合物的方法。

2. 发明授权

US06243980B1 Protease inhibitor assay 失效
标题翻译：蛋白酶抑制剂测定
公开(公告)号：US06243980B1
公开(公告)日：2001-06-12
申请号：US09035820
申请日：1998-03-06
申请人： Irena Bronstein , John Voyta , Michelle Palmer , Bonnie Tillotson
发明人： Irena Bronstein , John Voyta , Michelle Palmer , Bonnie Tillotson
IPC分类号： G01N33533
CPC分类号： G01N33/582 , C12Q1/37 , G01N33/581 , G01N2333/81 , Y10S436/80 , Y10S436/805
摘要： Heterogenous and homogenous assays are provided for the detection of protease inhibitory activity in a sample or target compound, taking advantage of the chemiluminescent characteristics of 1,2-dioxetanes. In the heterogenous assay, a peptide bearing a cleavage site for the protease of interest is provided with a first member of a first ligand binding pair at one end, and a first member of a second ligand binding pair at the other end. The other member of the first ligand binding pair is attached to a surface, which binds the peptide, or protease substrate, to the surface. The peptide substrate is combined with the protease and target compound or sample. Substrate cleavage, if not inhibited, is allowed to occur, and any unbound cleaved fragments are removed. An enzyme complexed with the second member of the second ligand binding pair is added, and allowed to bind to any of the (uncleaved) first member of the second ligand binding pair remaining. Unbound complex is removed, and a 1,2-dioxetane substrate for the enzyme is added. If any peptide substrate has not been cleaved, the dioxetane will chemiluminesce, indicating inhibitory activity. In a homogenous assay, the same substrate bears at one end a fluorescent energy accepting moiety, and at the other end a 1,2-dioxetane or precursor. If the substrate is cleaved by the protease, the dioxetane and the fluorescent moiety are not in close physical relationship, and no energy transfer occurs when the dioxetane is caused to decompose. If cleavage has not occurred, indicating inhibition, when the dioxetane is caused to decompose, energy is transferred to the fluorescing entity, which releases light of a wavelength recognizably distinct from that of the dioxetane.
摘要翻译：提供异源和均一的测定法，用于检测样品或目标化合物中蛋白酶抑制活性，利用1,2-二氧杂环己烷的化学发光特性。在异源测定中，具有目的蛋白酶的切割位点的肽在一端具有第一配体结合对的第一个成员，另一端具有第二配体结合对的第一个成员。第一配体结合对的另一个成员连接到表面，其将肽或蛋白酶底物结合到表面。将肽底物与蛋白酶和目标化合物或样品组合。如果不抑制底物裂解，则可以发生底物裂解，并且去除任何未结合的切割的片段。加入与第二配体结合对的第二个成员配合的酶，并使其与残留的第二配体结合对的任何（未切割的）第一个成员结合。去除未结合的络合物，并加入酶的1,2-二氧环乙烷底物。如果任何肽底物未被切割，二氧杂环丁烷将化学发光，表明抑制活性。在同质测定中，相同的底物在一端承载荧光能量接受部分，另一端在1,2-二氧杂环丁烷或前体上。如果底物被蛋白酶切割，二氧杂环丁烷和荧光部分不是很密切的物理关系，当二氧杂环丁烷分解时，不会发生能量转移。如果没有发生裂解，表明抑制，当二氧杂环丁烷分解时，能量被转移到荧光实体，其释放与二氧杂环丁烷的波长可辨别的波长的光。

3. 发明授权

US07235374B2 Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation 有权
标题翻译：使用报告酶突变体互补对G蛋白偶联受体和孤儿受体功能敏感检测的系统
公开(公告)号：US07235374B2
公开(公告)日：2007-06-26
申请号：US10959611
申请日：2004-10-05
申请人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
发明人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
IPC分类号： C07K14/705 , C07K19/00 , C12N15/62 , G01N33/566
CPC分类号： G01N33/566 , G01N2333/726 , G01N2500/10
摘要： Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.
摘要翻译：检测G蛋白偶联受体（GPCR）活性的方法; 测定GPCR活性的方法; 并描述了筛选GPCR配体，G蛋白偶联受体激酶（GRK）活性和与GPCR调节过程的组分相互作用的化合物的方法。包括用于扩展用于测定GPCR活性的ICAST技术的方法，用于配体捕鱼和激动剂或拮抗剂筛选。这些方法包括：将已知或孤儿GPCR开放阅读框中的氨基酸/苏氨酸磷酸化位点工程化，以增加抑制蛋白对GPCR活化形式的亲和力或增加抑制蛋白在激活的GPCR上的停留时间; 在不存在可能是限制性的G蛋白偶联受体激酶的情况下结合激活的GPCR的工程化突变抑制蛋白; 和具有增加对具有或不具有磷酸化作用的活化GPCR的亲和力的工程突变超级抑制蛋白。这些方法旨在增加GPCR / ICAST技术在G蛋白偶联受体激酶不存在或限制的情况下的稳健性，或其中GPCR不能有效下调或快速复敏（因此具有不稳定相互作用与arrestin）。包括也是使用ICAST互补酶片段监测GPCR同源和异二聚化的更具体的方法，用于药物铅发现和孤儿GPCR的配体和功能发现的应用。

4. 发明申请

US20060040250A1 Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation 有权
公开(公告)号：US20060040250A1
公开(公告)日：2006-02-23
申请号：US10959611
申请日：2004-10-05
申请人： Michelle Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
发明人： Michelle Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
IPC分类号： C12Q1/00 , G01N33/567 , G01N33/53
CPC分类号： G01N33/566 , G01N2333/726 , G01N2500/10
摘要： Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.

5. 发明授权

US06800445B2 Systems for sensitive detection of G-protein coupled receptor and orphan receptor function using reporter enzyme mutant complementation 失效
标题翻译：使用报告酶突变体互补对G蛋白偶联受体和孤儿受体功能敏感检测的系统
公开(公告)号：US06800445B2
公开(公告)日：2004-10-05
申请号：US09759152
申请日：2001-01-16
申请人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
发明人： Michelle A. J. Palmer , Melissa Gee , Bonnie Tillotson , Xiao-jia Chang
IPC分类号： G01N33567
CPC分类号： G01N33/566 , G01N2333/726 , G01N2500/10
摘要： Methods for detecting G-protein coupled receptor (GPCR) activity; methods for assaying GPCR activity; and methods for screening for GPCR ligands, G-protein-coupled receptor kinase (GRK) activity, and compounds that interact with components of the GPCR regulatory process are described. Included are methods for expanding ICAST technologies for assaying GPCR activity with applications for ligand fishing, and agonist or antagonist screening. These methods include: engineering seronine/threonine phosphorylation sites into known or orphan GPCR open reading frames in order to increase the affinity of arrestin for the activated form of the GPCR or to increase the reside time of arrestin on the activated GPCR; engineering mutant arrestin proteins that bind to activated GPCRs in the absence of G-protein coupled receptor kinases which may be limiting; and engineering mutant super arrestin proteins that have an increased affinity for activated GPCRs with or without phosphorylation. These methods are intended to increase the robustness of the GPCR/ICAST technology in situations in which G-protein coupled receptor kinases are absent or limiting, or in which the GPCR is not efficiently down-regulated or is rapidly resensitized (thus having a labile interaction with arrestin). Included are also more specific methods for using ICAST complementary enzyme fragments to monitor GPCR homo- and hetero-dimerization with applications for drug lead discovery and ligand and function discovery for orphan GPCRs.
摘要翻译：检测G蛋白偶联受体（GPCR）活性的方法; 测定GPCR活性的方法; 并描述了筛选GPCR配体，G蛋白偶联受体激酶（GRK）活性和与GPCR调节过程的组分相互作用的化合物的方法。包括用于扩展用于测定GPCR活性的ICAST技术的方法，用于配体捕鱼和激动剂或拮抗剂筛选。这些方法包括：将已知或孤儿GPCR开放阅读框中的氨基酸/苏氨酸磷酸化位点工程化，以增加抑制蛋白对GPCR活化形式的亲和力或增加抑制蛋白在激活的GPCR上的停留时间; 在不存在可能是限制性的G蛋白偶联受体激酶的情况下结合激活的GPCR的工程化突变抑制蛋白; 和具有增加对具有或不具有磷酸化作用的活化GPCR的亲和力的工程突变超级抑制蛋白。这些方法旨在增加GPCR / ICAST技术在G蛋白偶联受体激酶不存在或限制的情况下的稳健性，或其中GPCR不能有效下调或快速复敏（因此具有不稳定相互作用与arrestin）。包括也是使用ICAST互补酶片段监测GPCR同源和异二聚化的更具体的方法，用于药物铅发现和孤儿GPCR的配体和功能发现的应用。

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