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1.发明授权Protein fragment complementation assays for high-throughput and high-content screening 失效用于高通量和高含量筛选的蛋白质片段互补测定公开(公告)号：US07935493B2
公开(公告)日：2011-05-03
申请号：US11450379
申请日：2006-06-12
申请人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
发明人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
IPC分类号： G01N33/53 , G01N33/532 , G01N33/533 , G01N33/573 , G01N21/00 , G01N21/76 , C07K14/00
CPC分类号： G01N33/5011 , G01N33/5008 , G01N33/5041 , G01N33/542 , G01N33/6845 , G01N2500/02 , Y02A90/26
摘要： The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. Single-color and multi-color assays are disclosed. Further disclosed are universal expression vectors with cassettes that allow the rapid construction of assays for a large and diverse number of gene/reporter combinations. The development of such assays is shown to be straightforward, providing for a broad, flexible and biologically relevant platform for drug discovery.
摘要翻译： 本发明提供用于药物发现的蛋白质片段互补测定法，特别是鉴定激活或抑制细胞途径的化合物。 基于与适当的PCA报道子组合的相互作用的蛋白质对的选择，测定可以以高通量或高含量模式进行，并且可以用于化合物文库的自动化筛选。 可以通过cDNA文库筛选来选择相互作用的对; 通过基因相互作用作图; 或通过路径的先验知识。 可以使用本文提供的方法构建荧光和发光测定。 针对多种报道者描述了适合于高通量或高含量（高上下文）测定形式的PCA报告人的选择，具体提供了单体酶和荧光蛋白的实例。 描述了用于构建生物化学途径中的一个或多个步骤的这种测定方法; 测试来自组合，天然产物，肽，抗体，核酸或其他不同文库的化合物对感兴趣的蛋白质或途径的影响; 并使用筛选结果来鉴定激活或抑制感兴趣的蛋白质或途径的特定化合物。 公开了单色和多色测定。 进一步公开的是具有盒的通用表达载体，其允许快速构建用于大量和多样化数量的基因/报道子组合的测定。 显示出这种测定的发展是直接的，为药物发现提供了广泛，灵活和生物学上相关的平台。
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2.发明申请Protein fragment complementation assays for high-throughput and high-content screening 审中-公开用于高通量和高含量筛选的蛋白质片段互补测定公开(公告)号：US20120149597A1
公开(公告)日：2012-06-14
申请号：US13067007
申请日：2011-05-02
申请人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
发明人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
CPC分类号： G01N33/5011 , G01N33/5008 , G01N33/5041 , G01N33/542 , G01N33/6845 , G01N2500/02 , Y02A90/26
摘要： The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. Single-color and multi-color assays are disclosed. Further disclosed are universal expression vectors with cassettes that allow the rapid construction of assays for a large and diverse number of gene/reporter combinations. The development of such assays is shown to be straightforward, providing for a broad, flexible and biologically relevant platform for drug discovery.
摘要翻译： 本发明提供用于药物发现的蛋白质片段互补测定法，特别是鉴定激活或抑制细胞途径的化合物。 基于与适当的PCA报道子组合的相互作用的蛋白质对的选择，测定可以以高通量或高含量模式进行，并且可以用于化合物文库的自动化筛选。 可以通过cDNA文库筛选来选择相互作用的对; 通过基因相互作用作图; 或通过路径的先验知识。 可以使用本文提供的方法构建荧光和发光测定。 针对多种报道者描述了适合于高通量或高含量（高上下文）测定形式的PCA报告人的选择，具体提供了单体酶和荧光蛋白的实例。 描述了用于构建生物化学途径中的一个或多个步骤的这种测定方法; 测试来自组合，天然产物，肽，抗体，核酸或其他不同文库的化合物对感兴趣的蛋白质或途径的影响; 并使用筛选结果来鉴定激活或抑制感兴趣的蛋白质或途径的特定化合物。 公开了单色和多色测定。 进一步公开的是具有盒的通用表达载体，其允许快速构建用于大量和多样化数量的基因/报道子组合的测定。 显示出这种测定的发展是直接的，为药物发现提供了广泛，灵活和生物相关的平台。
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3.发明授权In vivo screening of protein-protein interactions with protein-fragment complementation assays 失效蛋白质 - 蛋白质相互作用与蛋白质片段互补测定的体内筛选公开(公告)号：US07855167B2
公开(公告)日：2010-12-21
申请号：US10728355
申请日：2003-12-05
CPC分类号： G01N33/542 , C07K14/43595 , G01N33/58 , G01N33/68 , G01N2800/52
摘要： The present invention describes rapid methods to screen for biomolecular interactions in vivo based on protein fragment complementation assays (PCA). We have demonstrated an in vivo library-versus-library screening strategy that has numerous applications in the identification of novel protein-protein interactions and in directed evolution. Also we demonstrate the detection of protein-protein interactions starting with defined (full-length) cDNAs, and the concomitant generation of functional assays that provide initial validation of the cDNA products as being biologically relevant. Also, we screened a large cDNA collection using automated PCA, combined with quantitative detection of protein-protein complexes. The invention enables bait-vs.-library, library-vs.-library and defined gene screening in any type of cell or cellular context, and using a wide range of reporters and detection methods. The invention allows for identifying and validating genes involved in any cellular process and also provide assays to study effects of potential drugs, or gene knockouts on specific pathways.
摘要翻译： 本发明描述了基于蛋白质片段互补测定（PCA）在体内筛选生物分子相互作用的快速方法。 我们已经展示了体内文库对文库筛选策略，其在鉴定新的蛋白质 - 蛋白质相互作用和定向进化中具有许多应用。 我们还展示了从定义的（全长）cDNA开始的蛋白质 - 蛋白质相互作用的检测，以及伴随产生的功能测定，提供cDNA产物的初步验证是生物相关的。 此外，我们使用自动PCA筛选了大量cDNA集合，结合蛋白质 - 蛋白质复合物的定量检测。 本发明使得在任何类型的细胞或细胞上下文中使用诱饵与文库，文库与文库以及定义的基因筛选，以及使用广泛的记者和检测方法。 本发明允许鉴定和验证参与任何细胞过程的基因，并且还提供测定以研究潜在药物或基因敲除对特定途径的影响。
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4.发明授权Protein fragment complementation assays for high-throughput and high-content screening 失效用于高通量和高含量筛选的蛋白质片段互补测定公开(公告)号：US07062219B2
公开(公告)日：2006-06-13
申请号：US10772021
申请日：2004-02-05
申请人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
发明人： Stephen William Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John K. Westwick
CPC分类号： G01N33/5011 , G01N33/5008 , G01N33/5041 , G01N33/542 , G01N33/6845 , G01N2500/02 , Y02A90/26
摘要： The present invention provides protein single-color and multi-color protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter such as monomeric enzymes and fluorescent proteins, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. The development of such assays provides for a broad, flexible and biologically relevant platform for drug discovery.
摘要翻译： 本发明提供用于药物发现的蛋白质单色和多色蛋白质片段互补测定法，特别是鉴定激活或抑制细胞途径的化合物。 基于与适当的PCA报告物如单体酶和荧光蛋白结合的相互作用的蛋白质对的选择，测定可以以高通量或高含量模式进行，并且可以用于化合物文库的自动筛选。 描述了用于构建生物化学途径中的一个或多个步骤的这种测定方法; 测试来自组合，天然产物，肽，抗体，核酸或其他不同文库的化合物对感兴趣的蛋白质或途径的影响; 并使用筛选结果来鉴定激活或抑制感兴趣的蛋白质或途径的特定化合物。 这种测定的开发提供了用于药物发现的广泛，灵活和生物相关的平台。
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5.发明授权公开(公告)号：US08101364B2
公开(公告)日：2012-01-24
申请号：US11656543
申请日：2007-01-23
IPC分类号： G01N33/53
CPC分类号： C07K14/43595 , G01N33/542 , G01N33/58 , G01N33/582 , G01N33/68 , G01N33/6845 , G01N2333/43595
摘要： The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP The invention also encompasses red fluorescent PCAs based on Discosoma red fluorescent protein (RFP PCA) and a kindling fluorescent protein PCA (KFP1 PCA) derived from Anemonia sulcata. Any useful mutation of a fluorescent protein can be engineered into a fragment, generating a wide range of assays useful for drug discovery, target validation, high-throughput screening, high-content screening, pathway mapping, drug mechanism-of-action studies, biosensors, and diagnostics.
摘要翻译： 本发明涉及基于荧光蛋白的蛋白质片段互补测定（PCA）和测定组合物。 本发明提供了分离荧光蛋白并产生具有PCA所需光谱特征的突变片段的方法。 本发明包括基于来自Aequorea，Anemonia和Anthozoa的荧光蛋白的测定和组合物。 特别地，本发明涉及具有比野生型蛋白质更好的光谱性质的突变荧光蛋白的片段。 本发明包括A.维多利亚绿色荧光蛋白（GFP），特别是黄色荧光蛋白（EYFP和超EYFP），“维纳斯”，青色“黄水晶”，蓝色，青绿色和可光活化变体的突变体版本的片段 的GFP本发明还包括基于Discosoma红色荧光蛋白（RFP PCA）的红色荧光PCA和源自Anemonia sulcata的点燃荧光蛋白PCA（KFP1 PCA）。 荧光蛋白的任何有用的突变可被工程化成片段，产生广泛的用于药物发现，靶标验证，高通量筛选，高含量筛选，途径作图，药物作用机理研究，生物传感器 ，和诊断。
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6.发明授权公开(公告)号：US07166424B2
公开(公告)日：2007-01-23
申请号：US10724178
申请日：2003-12-01
CPC分类号： C07K14/43595 , G01N33/542 , G01N33/58 , G01N33/582 , G01N33/68 , G01N33/6845 , G01N2333/43595
摘要： The present invention is directed to Protein-fragment Complementation Assays (PCAs) and assay compositions based on fluorescent proteins. The invention provides methods for fragmenting fluorescent proteins and generating mutant fragments with desired spectral characteristics for PCA. The invention encompasses assays and compositions based on fluorescent proteins from the species Aequorea, Anemonia and Anthozoa. In particular, the invention is directed to fragments of mutant fluorescent proteins having improved spectral properties over the wild-type proteins. The invention encompasses fragments of mutant versions of A. Victoria green fluorescent protein (GFP), in particular yellow fluorescent proteins (EYFP and super-EYFP), ‘Venus’, cyan, ‘citrine’, blue, cyan-green, and photoactivatable variants of GFP.
摘要翻译： 本发明涉及基于荧光蛋白的蛋白质片段互补测定（PCA）和测定组合物。 本发明提供了分离荧光蛋白并产生具有PCA所需光谱特征的突变片段的方法。 本发明包括基于来自Aequorea，Anemonia和Anthozoa的荧光蛋白的测定和组合物。 特别地，本发明涉及具有比野生型蛋白质更好的光谱性质的突变荧光蛋白的片段。 本发明包括A.维多利亚绿色荧光蛋白（GFP），特别是黄色荧光蛋白（EYFP和超EYFP），“维纳斯”，青色“黄水晶”，蓝色，青绿色和可光活化变体的突变体版本的片段 的GFP。
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7.发明申请Protein fragment complementation assays for high-throughput and high-content screening 失效用于高通量和高含量筛选的蛋白质片段互补测定公开(公告)号：US20060224331A1
公开(公告)日：2006-10-05
申请号：US11450379
申请日：2006-06-12
申请人： Stephen Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John Westwick
发明人： Stephen Watson Michnick , Ingrid Remy , Marnie MacDonald , Jane Lamerdin , Helen Yu , John Westwick
CPC分类号： G01N33/5011 , G01N33/5008 , G01N33/5041 , G01N33/542 , G01N33/6845 , G01N2500/02 , Y02A90/26
摘要： The present invention provides protein fragment complementation assays for drug discovery, in particular to identify compounds that activate or inhibit cellular pathways. Based on the selection of an interacting protein pair combined with an appropriate PCA reporter, the assays may be run in high-throughput or high-content mode and may be used in automated screening of libraries of compounds. The interacting pair may be selected by cDNA library screening; by gene-by-gene interaction mapping; or by prior knowledge of a pathway. Fluorescent and luminescent assays can be constructed using the methods provided herein. The selection of suitable PCA reporters for high-throughput or high-content (high-context) assay formats is described for a diversity of reporters, with particular detail provided for examples of monomeric enzymes and fluorescent proteins. Methods are described for constructing such assays for one or more steps in a biochemical pathway; testing the effects of compounds from combinatorial, natural product, peptide, antibody, nucleic acid or other diverse libraries on the protein or pathway(s) of interest; and using the results of the screening to identify specific compounds that activate or inhibit the protein or pathway(s) of interest. Single-color and multi-color assays are disclosed. Further disclosed are universal expression vectors with cassettes that allow the rapid construction of assays for a large and diverse number of gene/reporter combinations. The development of such assays is shown to be straightforward, providing for a broad, flexible and biologically relevant platform for drug discovery.
摘要翻译： 本发明提供用于药物发现的蛋白质片段互补测定法，特别是鉴定激活或抑制细胞途径的化合物。 基于与适当的PCA报道子组合的相互作用的蛋白质对的选择，测定可以以高通量或高含量模式进行，并且可以用于化合物文库的自动化筛选。 可以通过cDNA文库筛选来选择相互作用的对; 通过基因相互作用作图; 或通过路径的先验知识。 可以使用本文提供的方法构建荧光和发光测定。 针对多种报道者描述了适合于高通量或高含量（高上下文）测定形式的PCA报告人的选择，具体提供了单体酶和荧光蛋白的实例。 描述了用于构建生物化学途径中的一个或多个步骤的这种测定方法; 测试来自组合，天然产物，肽，抗体，核酸或其他不同文库的化合物对感兴趣的蛋白质或途径的影响; 并使用筛选结果来鉴定激活或抑制感兴趣的蛋白质或途径的特定化合物。 公开了单色和多色测定。 进一步公开的是具有盒的通用表达载体，其允许快速构建用于大量和多样化数量的基因/报道子组合的测定。 显示出这种测定的发展是直接的，为药物发现提供了广泛，灵活和生物相关的平台。
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8.发明申请High-content and high throughput assays for identification of lipid-regulating pathways, and novel therapeutic agents for lipid disorders 审中-公开用于鉴定脂质调节途径的高含量和高通量测定，以及用于脂质疾病的新型治疗剂公开(公告)号：US20100081632A1
公开(公告)日：2010-04-01
申请号：US12382066
申请日：2009-03-06
IPC分类号： A61K31/69 , G01N33/53 , G01N33/573 , C40B30/04 , A61K31/35
CPC分类号： G01N33/6845 , G01N33/542 , G01N33/92 , G01N2333/96411 , G01N2500/02
摘要： A method of assaying protein-protein interactions associated with proteins involved in lipid pathways using a protein fragment complementation assays, said method comprising the steps of: (a) identifying protein molecules that interact with said protein associated with lipid pathways; (b) selecting a protein reporter molecule; (c) effecting fragmentation of said protein reporter molecule such that said fragmentation results in reversible loss of reporter function; (d) fusing or attaching fragments of said protein reporter molecule separately to said interacting protein molecules as defined in step (a); (e) transfecting cells with nucleic acid constructs coding for the products of step (d); (f) reassociating said reporter fragments through interactions of the protein molecules that are fused or attached to said fragments; and (g) measuring directly or Indirectly the activity of said reporter molecule resulting from the reassociation of said reporter fragments.
摘要翻译： 一种使用蛋白质片段互补测定法测定涉及脂质途径的蛋白质相关的蛋白质 - 蛋白质相互作用的方法，所述方法包括以下步骤：（a）鉴定与所述与脂质途径相关的蛋白质相互作用的蛋白质分子; （b）选择蛋白质报告分子; （c）使所述蛋白质报告分子进行片段化，使得所述片段化导致报告基因功能的可逆损失; （d）将所述蛋白质报告分子的片段与步骤（a）中定义的所述相互作用蛋白分子分开融合或附着; （e）用编码步骤（d）的产物的核酸构建体转染细胞; （f）通过与所述片段融合或连接的蛋白质分子的相互作用使所述报道片段重新结合; 和（g）直接或间接测量所述报告分子的活性，所述活性由所述报道片段的重新连接而产生。
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公开(公告)号：US20060160109A1
公开(公告)日：2006-07-20
申请号：US11282745
申请日：2005-11-21
CPC分类号： G01N33/5008 , B82Y10/00 , B82Y30/00 , C12Q1/025 , G01N33/5014 , G01N33/5041
摘要： This invention provides principles, methods and compositions for ascertaining the mechanism of action of pharmacologically important compounds in the context of network biology, across the entire scope of the complex pathways of living cells. Importantly, the principles, methods and compositions provided allow a rapid assessment of the on-pathway and off-pathway effects of lead compounds and drug candidates in living cells, and comparisons of lead compounds with well-characterized drugs and toxicants to identify patterns associated with efficacy and toxicity. The invention will be useful in improving the drug discovery process, in particular by identifying drug leads with desired safety and efficacy and in effecting early attrition of compounds with potential adverse effects in man.
摘要翻译： 本发明提供了在网络生物学背景下确定药物重要化合物在活细胞复合途径的整个范围内的作用机制的原理，方法和组合物。 重要的是，提供的原理，方法和组合可以快速评估铅化合物和药物候选物在活细胞中的通路和脱离途径作用，以及铅化合物与良好表征的药物和毒物的比较，以鉴定与 功效和毒性。 本发明将有助于改进药物发现过程，特别是通过鉴定具有期望的安全性和功效的药物引线并且实现具有潜在副作用的化合物的早期损耗。
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公开(公告)号：US20060094682A1
公开(公告)日：2006-05-04
申请号：US11260164
申请日：2005-10-28
申请人： John Westwick , Jane Lamerdin , Stephen Owens , Marnie MacDonald
发明人： John Westwick , Jane Lamerdin , Stephen Owens , Marnie MacDonald
IPC分类号： A61K48/00 , A61K38/54 , A61K39/395
CPC分类号： C12Q1/485 , G01N33/502 , G01N33/6893 , G01N2500/00
摘要： The present invention discloses a method of treating an individual or animal with diabetes and/or obesity. The method comprises administering to the individual or animal a therapeutically effective amount of a protein tyrosine kinase inhibitor. Preferably, the preventative and therapeutic methods of the present invention involve administering—to a mammal in need thereof—a therapeutically effective amount of an inhibitor of a c-Src-family protein tyrosine kinase. The invention pertains to pharmaceutical compositions containing an inhibitor of a c-Src-family protein tyrosine kinase or an analog or metabolite thereof, or an inhibitor of another protein tyrosine kinase, and a pharmaceutically acceptable carrier. Purines and pyrimidines and other molecules useful in the treatment of diabetes and obesity are provided herein, in particular, pyrazolopyrimidines, cyanoquinolines, phenylaminopyrimidines, anilinoquinazolines and related compounds. The invention also provides cellular targets and assay compositions useful for the identification of additional novel therapeutic agents for the treatment of these disorders.
摘要翻译： 本发明公开了一种用糖尿病和/或肥胖症治疗个体或动物的方法。 该方法包括向个体或动物施用治疗有效量的蛋白酪氨酸激酶抑制剂。 优选地，本发明的预防和治疗方法包括向有需要的哺乳动物施用治疗有效量的c-Src家族蛋白酪氨酸激酶的抑制剂。 本发明涉及含有c-Src家族蛋白酪氨酸激酶或其类似物或代谢物的抑制剂或其它蛋白酪氨酸激酶的抑制剂和药学上可接受的载体的药物组合物。 本文提供了可用于治疗糖尿病和肥胖症的嘌呤和嘧啶和其它分子，特别是吡唑并嘧啶，氰基喹啉，苯基氨基嘧啶，苯胺基喹唑啉和相关化合物。 本发明还提供用于鉴定用于治疗这些疾病的其它新型治疗剂的细胞靶和测定组合物。
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