Development of a HTS-assay for inhibitors of a type 2C protein phosphatase (PPM

开发 2C 型蛋白磷酸酶抑制剂 (PPM) 的 HTS 测定法

• 批准号: 7993354
• 负责人: RICHARD E HONKANEN
• 金额: $ 14.83万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993354
• 关键词: Affect; Apoptosis; Biological; Biological Assay; Biology; Cells; Communities; Complex; Detection; Development; End Point Assay; Environment; Enzymes; Family; Gene Family; Growth Factor; Hormones; Human; Lead; Malignant Neoplasms; Mammals; Measures; Medical; Metals; Methods; Molecular Bank; Okadaic Acid; Oncogenes; Pathology; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Production; Protein Kinase; Protein p53; Protein phosphatase; Proteins; Reaction; Research; Role; Serine; Signal Transduction; Specificity; Stimulus; TP53 gene; Threonine; Tumor Suppressor Proteins; Tyrosine; base; calyculin A; cell growth; cell type; high throughput screening; human disease; inhibitor/antagonist; inorganic phosphate; mutant; protein phosphatase 2C; public health relevance; senescence; small molecule; tool

DESCRIPTION (provided by applicant): Human cells are constantly exposed to a variety of hormones, growth factors and other agents that affect cell growth. To integrate and interpret these external stimuli, complex signaling networks have evolved, which allow different types of cells to respond appropriately to their environment. In all mammals the reversible phosphorylation of proteins regulates many intracellular signaling networks that control cell growth, differentiation, senescence and programmed cell death (apoptosis). Protein phosphorylation occurs principally on serine, threonine and tyrosine residues, and the phosphorylation reaction is catalyzed by a large family of protein kinases. To date many compounds that function as potent and highly selective inhibitors of "key" protein kinases have been identified, and these inhibitors have proven to be powerful tools to probe the biology and pathology associated with the actions of protein kinases. In contrast, much less is known about the biology of protein phosphates. Notably the biological roles and pathology associated with phosphates belonging to the PP2C subfamily are poorly understood. To a large extent, this is due to the lack of probes. Unlike dual specificity and tyrosine phosphates, where the Cys-based catalytic mechanism allows for the development of substrate trapping mutants, the metal-based catalytic mechanism of PP2C-enzymes cannot be modified to produce substrate-trapping mutants. In addition, natural compounds that potently act on PPP-family phosphates (i.e. okadaic acid and calyculin A), do not affect PP2C activity. Therefore, tools to study PP2C-family phosphates are particularly desired by the research community. Having developed methods to produce a large amount of catalytically active PP2C4 (a known oncogene) and a fluorescent assay to reliably measure PP2C4 activity, the objective of this application is to develop a high throughput ready assay that can be used to identify specific, or highly selective, inhibitors for this biologically important phosphatase. The compounds produced from this effort will serve as powerful small molecule probes that will greatly aid efforts to elucidate the roles of PP2C4 in normal biology and human disease. They may also serve as lead compounds for the development of new drugs for medical management of human cancer. PUBLIC HEALTH RELEVANCE: Serine/threonine protein phosphatase 2C delta (PP2C4) has emerged as a regulator of p53 signaling networks and appears to act as an oncogene. Having developed methods to produce large amounts of (PP2C4) and an assay to measure (PP2C4) activity, we proposal is to identify a specific or highly selective inhibitor by developing methods that will enable a large scale screen in conjunction with the Molecular Libraries Probe Production Centers Network (MLPCN). The compounds produced from this effort will serve as powerful tool to probe the biology and pathology associated with (PP2C4).

描述(申请人提供)：人类细胞不断地暴露在各种激素、生长因子和其他影响细胞生长的因素中。为了整合和解释这些外部刺激，复杂的信号网络已经进化出来，允许不同类型的细胞对环境做出适当的反应。在所有哺乳动物中，蛋白质的可逆磷酸化调节许多细胞内信号网络，这些信号网络控制细胞的生长、分化、衰老和细胞程序性死亡(细胞凋亡)。蛋白质的磷酸化主要发生在丝氨酸、苏氨酸和酪氨酸残基上，而磷酸化反应是由一大类蛋白激酶催化的。到目前为止，已经发现了许多化合物，它们是“关键”蛋白激酶的有效和高选择性的抑制剂，这些抑制剂已被证明是探索与蛋白激酶作用相关的生物学和病理学的有力工具。相比之下，人们对蛋白质磷酸盐的生物学知之甚少。值得注意的是，与属于PP2C亚家族的磷酸盐相关的生物学作用和病理学还知之甚少。这在很大程度上是因为缺乏探头。与双特异性和酪氨酸磷酸酯不同，基于半胱氨酸的催化机制允许开发底物捕获突变体，而PP2C-酶的金属催化机制不能被修改以产生底物捕获突变体。此外，有效作用于PPP家族磷酸盐的天然化合物(即冈田酸和帽菌素A)不会影响PP2C的活性。因此，研究PP2C家族磷酸盐的工具尤其受到研究界的欢迎。在开发了大量生产具有催化活性的PP2C4(已知的癌基因)的方法和可靠地测量PP2C4活性的荧光分析方法之后，这一应用的目标是开发一种高通量的准备好的分析方法，可以用于识别这种具有生物重要性的磷酸酶的特异性或高选择性的抑制剂。通过这一努力产生的化合物将成为强大的小分子探针，将极大地有助于阐明PP2C4在正常生物学和人类疾病中的作用。它们还可以作为先导化合物，用于开发治疗人类癌症的新药。 公共卫生相关性：丝氨酸/苏氨酸蛋白磷酸酶2C增量(PP2C4)已成为p53信号网络的调节因子，并似乎是癌基因。在开发了大量生产(PP2C4)的方法和测量(PP2C4)活性的方法后，我们建议通过开发能够与分子文库探针生产中心网络(MLPCN)一起进行大规模筛选的方法来识别特定或高选择性的抑制剂。通过这一努力产生的化合物将成为探索与(PP2C4)相关的生物学和病理学的强大工具。