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    In vivo library-versus-library selection of optimized protein-protein interactions
    1.
    发明申请
    In vivo library-versus-library selection of optimized protein-protein interactions 失效
    体内文库对文库选择优化的蛋白质 - 蛋白质相互作用

    公开(公告)号：US20050208577A1

    公开(公告)日：2005-09-22

    申请号：US11134253

    申请日：2005-05-23

    申请人： Stephen Michnick ,  Joelle Pelletier ,  Katja Arndt ,  Andreas Pluckthun

    发明人： Stephen Michnick ,  Joelle Pelletier ,  Katja Arndt ,  Andreas Pluckthun

    IPC分类号： C07K14/435 ,  C12N15/10 ,  C12Q1/32 ,  C12Q1/68 ,  C40B30/04 ,  G01N33/53 ,  G01N33/542 ,  G01N33/573 ,  G01N33/58 ,  G01N33/68

    CPC分类号： C40B30/04 ,  C07K14/43595 ,  C07K2319/00 ,  C12N15/1055 ,  C12Q1/32 ,  G01N33/542 ,  G01N33/6845 ,  Y02A90/26

    摘要： The present invention describes a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. It allows for the screening of a protein library against a second protein library, rather than against a single bait protein, and thus has numerous applications in the study of protein-protein interactions. Additionally, it allows for the application of different selection stringencies. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into E. coli. Interaction between the library polypeptides was required for reconstitution of the enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0×106 combinations, and selected a novel leucine zipper pair which may be appropriate for use in further in vivo heterodimerization strategies.

    摘要翻译： 本发明描述了快速和有效的体内文库对文库筛选策略，用于鉴定异构二聚化多肽的最佳相互作用对。 它允许针对第二蛋白质文库筛选蛋白质文库，而不是针对单个诱饵蛋白质，因此在蛋白质 - 蛋白质相互作用的研究中具有许多应用。 此外，它允许应用不同的选择严格性。 两个亮氨酸拉链文库在与疏水核心相邻的位置半随机化，与酶二氢叶酸还原酶（mDHFR）的两个设计的片段中的任一个遗传融合，并共转化到大肠杆菌中。 需要文库多肽之间的相互作用来重构mDHFR的酶活性，从而允许细菌生长。 所得菌落的分析揭示了相对于原始文库的拉链序列的重要偏倚，这与选择稳定的异源二聚体对一致。 使用更弱的关联mDHFR片段，我们增加了选择的严格性。 我们通过在液体培养中多次传代合并的选定菌落，丰富了表现最好的亮氨酸拉链对，因为最佳对允许更好的细菌繁殖。 这种竞争性增长允许放大对中的小差异，并且以不同的速率富集不同的序列位置。 我们将这些选择过程应用于2.0×10 6组合的文库对文库样品，并选择了可能适用于进一步体内异二聚化策略的新型亮氨酸拉链对。

    Protein fragment complementation assays for the detection of biological or drug interactions
    2.
    发明申请
    Protein fragment complementation assays for the detection of biological or drug interactions 失效
    用于检测生物或药物相互作用的蛋白质片段互补测定法

    公开(公告)号：US20070148682A1

    公开(公告)日：2007-06-28

    申请号：US11650466

    申请日：2007-01-08

    申请人： Stephen Michnick ,  Joelle Pelletier ,  Ingrid Remy

    发明人： Stephen Michnick ,  Joelle Pelletier ,  Ingrid Remy

    IPC分类号： C12Q1/68 ,  G06F19/00 ,  C12Q1/37

    CPC分类号： G01N33/6845 ,  A01K67/0271 ,  A01K2267/0331 ,  A01K2267/0393 ,  C07K14/43595 ,  C07K2319/00 ,  C12N15/1055 ,  G01N33/5008 ,  G01N33/542 ,  G01N33/573 ,  G01N33/58 ,  G01N33/581 ,  G01N33/68 ,  G01N33/6803 ,  Y02A90/26

    摘要： We describe a strategy for designing and implementing protein-fragment complementation assays (PCAs) to detect biomolecular interactions in vivo and in vitro. The design, implementation and broad applications of this strategy are illustrated with a large number of enzymes with particular detail provided for the example of murine dihydrofolate reductase (DHFR). Fusion peptides consisting of N- and C-terminal fragments of murine DHFR fused to GCN4 leucine zipper sequences were coexpressed in Escherichia coli grown in minimal medium, where the endogenous DHFR activity was inhibited with trimethoprim. Coexpression of the complementary fusion products restored colony formation. Survival only occurred when both DHFR fragments were present and contained leucine-zipper forming sequences, demonstrating that reconstitution of enzyme activity requires assistance of leucine zipper formation. DHFR fragment-interface point mutants of increasing severity (Ile to Val, Ala and Gly) resulted in a sequential increase in E. coli doubling times illustrating the successful DHFR fragment reassembly rather than non-specific interactions between fragments. This assay could be used to study equilibrium and kinetic aspects of molecular interactions including protein-protein, protein-DNA, protein-RNA, protein-carbohydrate and protein-small molecule interactions, for screening cDNA libraries for binding of a target protein with unknown proteins or libraries of small organic molecules for biological activity. The selection and design criteria applied here is developed for numerous examples of clonal selection, colorometric, fluorometric and other assays based on enzymes whose products can be measured. The development of such assay systems is shown to be simple, and provides for a diverse set of protein fragment complementation applications.

    摘要翻译： 我们描述了设计和实施蛋白质片段互补测定（PCAs）以检测体内和体外生物分子相互作用的策略。 该策略的设计，实现和广泛应用用大量酶进行了说明，具体细节提供了鼠二氢叶酸还原酶（DHFR）的例子。 由融合到GCN4亮氨酸拉链序列的鼠DHFR的N-和C-末端片段组成的融合肽在基本培养基中生长的大肠杆菌中共表达，其中内源性DHFR活性被甲氧苄啶抑制。 互补融合产物的共表达恢复了菌落形成。 存活只发生在两个DHFR片段存在并含有亮氨酸 - 拉链形成序列时，表明重组酶活性需要辅助亮氨酸拉链形成。 增加严重程度（Ile至Val，Ala和Gly）的DHFR片段 - 接口点突变体导致大肠杆菌倍增时间的顺序增加，说明成功的DHFR片段重组而不是片段之间的非特异性相互作用。 该测定可用于研究包括蛋白质蛋白质，蛋白质-DNA，蛋白质-RNA，蛋白质 - 碳水化合物和蛋白质 - 小分子相互作用在内的分子相互作用的平衡和动力学方面，用于筛选靶向蛋白质与未知蛋白质结合的cDNA文库 或用于生物活性的小有机分子的文库。 这里应用的选择和设计标准是针对克隆选择，色度，荧光测定和其他基于可以测量其产物的酶的测定的许多实例开发的。 这种测定系统的开发被证明是简单的，并且提供了多种蛋白质片段互补应用的集合。

    Mapping molecular interactions in plants with protein fragments complementation assays
    3.
    发明申请
    Mapping molecular interactions in plants with protein fragments complementation assays 审中-公开
    用蛋白质片段互补测定法绘制植物中的分子相互作用

    公开(公告)号：US20050255452A1

    公开(公告)日：2005-11-17

    申请号：US11090215

    申请日：2005-03-28

    申请人： Normand Brisson ,  Stephen Michnick

    发明人： Normand Brisson ,  Stephen Michnick

    IPC分类号： C07K14/435 ,  C12N15/10 ,  C12N15/82 ,  C12Q1/00 ,  C12Q1/68 ,  G01N33/569 ,  G01N33/58 ,  G01N33/68

    CPC分类号： G01N33/6845 ,  C07K14/43595 ,  C12N15/1055 ,  C12N15/8216 ,  C12N15/8242 ,  G01N33/56961 ,  G01N33/58 ,  G01N33/68 ,  Y02A90/26

    摘要： The present invention describes a method of expressing PCA interacting partners in plant material comprising: (A) transforming said material with: (1) a first construct coding for a first fusion product comprising (a) a first fragment of a first molecule whose fragments can exhibit a detectable activity when associated and (b) a first protein-protein interacting domain; and (2) a second construct coding for a second fusion product comprising (a) a second fragment of said first molecule and (b) a second protein-protein interacting domain that can bind (1)(b); (B) culturing said material under conditions allowing expression of said PCA interacting partners; and (C) detecting said activity.

    摘要翻译： 本发明描述了在植物材料中表达PCA相互作用配偶体的方法，其包括：（A）使用以下物质转化所述材料：（1）编码第一融合产物的第一构建体，其包含（a）第一分子的第一片段， 在相关联时呈现可检测活性，（b）第一蛋白 - 蛋白质相互作用结构域; （2）编码第二融合产物的第二构建体，其包含（a）所述第一分子的第二片段和（b）能结合（1）（b）的第二蛋白质 - 蛋白质相互作用结构域; （B）在允许表达所述PCA相互作用的伴侣的条件下培养所述材料; 和（C）检测所述活性。

    Methods for identifying new drug leads and new therapeutic uses for known drugs
    4.
    发明申请
    Methods for identifying new drug leads and new therapeutic uses for known drugs 审中-公开
    识别新药物的方法和已知药物的新治疗用途

    公开(公告)号：US20060094059A1

    公开(公告)日：2006-05-04

    申请号：US11230569

    申请日：2005-09-21

    申请人： John Westwick ,  Marnie MacDonald ,  Helen Yu ,  Stephen Owens ,  Stephen Michnick

    发明人： John Westwick ,  Marnie MacDonald ,  Helen Yu ,  Stephen Owens ,  Stephen Michnick

    IPC分类号： C40B40/10 ,  G01N33/53

    CPC分类号： G01N33/5005 ,  G01N33/5035 ,  G01N33/5041

    摘要： The screening system utilizes dynamic measurements of pathway activity to detect the activities of drugs within cellular pathways. The methods of the invention can be used to identify previously unknown drug activities and therapeutic uses, even for drugs that have been well characterized with standard biochemical assays. We demonstrated the utility of the invention by screening a portion of the known pharmacopeia. We identified dozens of drugs, previously or currently marked for a variety of indications, with surprising and previously-unsuspected activity against ‘hallmark’ cancer pathways. We also showed that over 20 of these drugs indeed have anti-proliferative activity in human tumor cells, underscoring the utility and predictability of the screening system. The methodology will extend the utility of the current pharmacopeia and provide the basis for de novo discovery of drugs with a broad range of therapeutic indications.

    摘要翻译： 筛选系统利用通路活性的动态测量来检测细胞通路内药物的活性。 本发明的方法可以用于鉴定以前未知的药物活性和治疗用途，即使对于通过标准生化测定法进行了充分表征的药物也是如此。 我们通过筛选一部分已知药典证明了本发明的效用。 我们确定了以前或目前标有各种适应症的数十种药物，对“标志”癌症途径感到惊奇和先前不知情的活动。 我们还显示，超过20种这些药物确实在人类肿瘤细胞中具有抗增殖活性，强调了筛选系统的效用和可预测性。 该方法将扩大目前药典的效用，为从头发现具有广泛治疗适应症的药物提供依据。

    Fragments of fluorescent proteins for protein fragment complementation assays
    5.
    发明申请
    Fragments of fluorescent proteins for protein fragment complementation assays 有权
    用于蛋白质片段互补测定的荧光蛋白片段

    公开(公告)号：US20070254373A1

    公开(公告)日：2007-11-01

    申请号：US11656543

    申请日：2007-01-23

    申请人： Stephen Michnick ,  Marnie MacDonald ,  Jane Lamerdin

    发明人： Stephen Michnick ,  Marnie MacDonald ,  Jane Lamerdin

    IPC分类号： G01N33/00 ,  C07K14/00

    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。 本发明提供了用于分解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。

    Harnessing network biology to improve drug discovery
    7.
    发明申请
    Harnessing network biology to improve drug discovery 审中-公开
    利用网络生物学改善药物发现

    公开(公告)号：US20060160109A1

    公开(公告)日：2006-07-20

    申请号：US11282745

    申请日：2005-11-21

    申请人： Marnie MacDonald ,  John Westwick ,  Brigitte Keon ,  Jane Lamerdin ,  Stephen Michnick

    发明人： Marnie MacDonald ,  John Westwick ,  Brigitte Keon ,  Jane Lamerdin ,  Stephen Michnick

    IPC分类号： C40B40/08 ,  C40B40/10

    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。

    Fluorescent labeling of specific protein targets in vitro and in vivo
    8.
    发明申请
    Fluorescent labeling of specific protein targets in vitro and in vivo 失效
    体外和体内特异性蛋白质靶标的荧光标记

    公开(公告)号：US20060147948A1

    公开(公告)日：2006-07-06

    申请号：US11153398

    申请日：2005-06-16

    申请人： Jeffrey Keillor ,  Stephen Michnick ,  Stephane Girouard

    发明人： Jeffrey Keillor ,  Stephen Michnick ,  Stephane Girouard

    IPC分类号： C12Q1/68 ,  C07D473/10 ,  C07D405/14 ,  C07D403/14

    CPC分类号： G01N33/582

    摘要： New methods are provided for the post-genomic era that will permit the analysis of the dynamic expression and localization of gene products in living cells. Herein we propose the development of such a method from a bioorganic approach involving organic synthesis and protein engineering. Specifically, novel compounds bearing two maleimide groups attached directly to fluorescent cores will be prepared, whose latent fluorescence is quenched until their maleimide groups undergo a specific thiol addition reaction. Complementary a-helical proteins are designed bearing two cysteine residues appropriately positioned to react with our novel fluorogens. Genetically fusing our helical probe peptides to test proteins of interest, we can selectively label the target sequence in living cells with our small synthetic fluorogenic molecules. The scope of this technique is described in the context of studying protein localization and protein-protein interactions in living cells.

    摘要翻译： 为基因组后时代提供了新的方法，可以分析活细胞中基因产物的动态表达和定位。 在这里，我们提出从涉及有机合成和蛋白质工程的生物有机方法开发这种方法。 具体地说，将制备直接连接到荧光核心上的具有两个马来酰亚胺基团的新型化合物，其潜伏荧光骤冷直到其马来酰亚胺基团进行特定的硫醇加成反应。 设计了互补的α-螺旋蛋白，其具有适当定位以与我们的新型氟原子反应的两个半胱氨酸残基。 基因上融合我们的螺旋探针肽来测试感兴趣的蛋白质，我们可以用我们的小合成荧光分子选择性地标记活细胞中的靶序列。 在研究活细胞中蛋白质定位和蛋白质 - 蛋白质相互作用的背景下描述了该技术的范围。