Triple threat screening for modifiers of Protein Ser/Thr Phosphatase 2C

蛋白 Ser/Thr 磷酸酶 2C 修饰物的三重威胁筛查

基本信息

  • 批准号:
    7555514
  • 负责人:
  • 金额:
    $ 15.15万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2008
  • 资助国家:
    美国
  • 起止时间:
    2008-06-15 至 2010-05-31
  • 项目状态:
    已结题

项目摘要

DESCRIPTION (provided by applicant): Summary Protein phosphatase PP2C encompasses a family of a dozen human enzymes with related sequences that fold into a unique 3D structure that exhibits metal ion dependence, a catalytic mechanism and substrate specificity distinct from other types of protein Ser/Thr phosphatases. The PP2C are fundamentally important, with multiple genes conserved from yeast to human, and gene knockout phenotypes implicate PP2C in control of responses to radiation damage and other forms of cellular stress. Transcription of the PP2C isoform known as Wip1 (Wild type p53 Induced Phosphatase; or PP2C or PPM1D) is controlled by the tumor suppressor p53. Deletion of this gene results in activation of the DNA damage response pathway and suppresses tumor formation by oncogenes. To date there are no potent synthetic or natural product small molecule inhibitors or activators for any PP2C, which has hampered investigations of their biological functions and prevented development of drugs that target this family of regulatory enzymes. This project proposes a triple threat approach, developing in parallel HTS-compatible assays for three different members of the PP2C family. The plan is to produce recombinant, purified phosphatases PP2C, PP2C and PP2C (Wip1) and establish optimum conditions for HTS assay using chromogenic and phosphopeptide substrates. This approach builds in secondary screening for selectivity and isoform specificity and has the potential to identify both broad spectrum and highly specific chemical modulators of PP2C activity. A second Aim is to develop and optimize a new In-Cell Western HTS assay that quantitates simultaneous staining with two different antibodies by dual wavelength infrared detection. This live cell assay will detect cell-permeable compounds that affect PP2C activity against endogenous substrates. The availability of small molecule modifiers would be a major advance that would benefit investigations of biological functions of different PP2C and pave the way for development of novel compounds with medicinal properties. Project Narrative Human physiology is governed by internal hormone signals and changes in the external environment. Cells respond to these signals through networks of protein kinase and phosphatase enzymes that dynamically switch on/off various processes by the addition and removal of phosphate groups on thousands of different proteins. Human diseases arise from imbalances between these kinases and phosphatases and new targeted therapies in clinical use are chemicals that block the activity of these enzymes. A family of phosphatase enzymes called PP2C regulates responses to DNA damage and cell stress. This project seeks to produce specific and dependable assays for PP2C to screen collections of thousands of chemicals and identify novel activators or inhibitors. Chemical modifiers of PP2C have potential medicinal applications and will be used as research tools to study signaling in normal and cancer cells.
描述(由申请人提供):概述蛋白磷酸酶PP 2C包括一个由十几种人类酶组成的家族,其相关序列折叠成独特的3D结构,表现出金属离子依赖性、催化机制和底物特异性,与其他类型的蛋白质Ser/Thr磷酸酶不同。PP 2C是非常重要的,从酵母到人类都有多个保守的基因,基因敲除表型表明PP 2C控制着对辐射损伤和其他形式的细胞应激的反应。被称为Wip 1(野生型p53诱导的磷酸酶;或PP 2C或PPM 1D)的PP 2C亚型的转录由肿瘤抑制因子p53控制。该基因的缺失导致DNA损伤反应途径的激活并抑制癌基因引起的肿瘤形成。到目前为止,还没有有效的合成或天然产物小分子抑制剂或激活剂的任何PP 2C,这阻碍了他们的生物学功能的研究,并阻止了药物的发展,目标是这个家庭的调节酶。该项目提出了一种三重威胁方法,为PP 2C家族的三个不同成员平行开发HTS兼容检测。该计划是生产重组,纯化的磷酸酶PP 2C,PP 2C和PP 2C(Wip 1),并建立最佳条件的HTS测定使用显色和磷酸肽底物。这种方法建立在二次筛选的选择性和亚型特异性,并有可能确定广谱和高度特异性的PP 2C活性的化学调节剂。第二个目的是开发和优化一种新的细胞内蛋白质HTS测定,该测定通过双波长红外检测定量两种不同抗体的同时染色。该活细胞试验将检测影响PP 2C对内源性底物活性的细胞渗透性化合物。小分子修饰剂的可用性将是一个重大进展,将有利于不同PP 2C的生物功能的研究,并为开发具有药用特性的新型化合物铺平道路。人体生理学受内部激素信号和外部环境变化的影响。细胞通过蛋白激酶和磷酸酶网络对这些信号做出反应,这些蛋白激酶和磷酸酶通过添加和去除数千种不同蛋白质上的磷酸基团来动态地打开/关闭各种过程。人类疾病是由这些激酶和磷酸酶之间的不平衡引起的,临床使用的新靶向疗法是阻断这些酶活性的化学品。一个称为PP 2C的磷酸酶家族调节对DNA损伤和细胞应激的反应。该项目旨在为PP 2C提供特异性和可靠的检测方法,以筛选数千种化学品的集合,并识别新型激活剂或抑制剂。PP 2C的化学修饰剂具有潜在的医学应用,并将被用作研究正常细胞和癌细胞中信号传导的研究工具。

项目成果

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DAVID L. BRAUTIGAN其他文献

DAVID L. BRAUTIGAN的其他文献

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{{ truncateString('DAVID L. BRAUTIGAN', 18)}}的其他基金

Phosphorylation & Function of Inhibitor-2
磷酸化
  • 批准号:
    7859325
  • 财政年份:
    2009
  • 资助金额:
    $ 15.15万
  • 项目类别:
Core--Microscopy
核心--显微镜
  • 批准号:
    7541724
  • 财政年份:
    2008
  • 资助金额:
    $ 15.15万
  • 项目类别:
Core--Microscopy
核心--显微镜
  • 批准号:
    7333212
  • 财政年份:
    2007
  • 资助金额:
    $ 15.15万
  • 项目类别:
Cell Signaling
细胞信号转导
  • 批准号:
    7304711
  • 财政年份:
    2006
  • 资助金额:
    $ 15.15万
  • 项目类别:
Core--Microscopy
核心--显微镜
  • 批准号:
    7312435
  • 财政年份:
    2006
  • 资助金额:
    $ 15.15万
  • 项目类别:
Reorganization of the Actin Cytoskeleton by Phosphatases
磷酸酶重组肌动蛋白细胞骨架
  • 批准号:
    7119328
  • 财政年份:
    2005
  • 资助金额:
    $ 15.15万
  • 项目类别:
Core--Microscopy
核心--显微镜
  • 批准号:
    6967722
  • 财政年份:
    2005
  • 资助金额:
    $ 15.15万
  • 项目类别:
CHROMIUM ENHANCEMENT OF INSULIN SIGNALING
铬增强胰岛素信号传导
  • 批准号:
    6657382
  • 财政年份:
    2002
  • 资助金额:
    $ 15.15万
  • 项目类别:
CHROMIUM ENHANCEMENT OF INSULIN SIGNALING
铬增强胰岛素信号传导
  • 批准号:
    7071883
  • 财政年份:
    2002
  • 资助金额:
    $ 15.15万
  • 项目类别:
CHROMIUM ENHANCEMENT OF INSULIN SIGNALING
铬增强胰岛素信号传导
  • 批准号:
    6747319
  • 财政年份:
    2002
  • 资助金额:
    $ 15.15万
  • 项目类别:

相似海外基金

Identification of Novel Protein Phosphatases in the ATM Signaling Pathway
ATM 信号通路中新型蛋白磷酸酶的鉴定
  • 批准号:
    8224187
  • 财政年份:
    2009
  • 资助金额:
    $ 15.15万
  • 项目类别:
Identification of Novel Protein Phosphatases in the ATM Signaling Pathway
ATM 信号通路中新型蛋白磷酸酶的鉴定
  • 批准号:
    7762036
  • 财政年份:
    2009
  • 资助金额:
    $ 15.15万
  • 项目类别:
Identification of Novel Protein Phosphatases in the ATM Signaling Pathway
ATM 信号通路中新型蛋白磷酸酶的鉴定
  • 批准号:
    7941711
  • 财政年份:
    2009
  • 资助金额:
    $ 15.15万
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