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; 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（由申请人提供）：人类细胞不断暴露于多种激素，生长因子和其他影响细胞生长的药物。为了整合和解释这些外部刺激，复杂的信号网络已经发展，这使不同类型的细胞能够适当响应其环境。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通信网络，这些网络控制细胞生长，分化，衰老和程序性细胞死亡（凋亡）。蛋白质磷酸化主要发生在丝氨酸，苏氨酸和酪氨酸残基上，磷酸化反应由大型蛋白激酶催化。迄今为止，已经确定了许多功能充当“关键”蛋白激酶有效且高度选择性抑制剂的化合物，并且这些抑制剂已被证明是探测与蛋白激酶作用相关的生物学和病理学的强大工具。相反，关于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知之甚少。在很大程度上，这是由于缺乏探针所致。与双重特异性和酪氨酸磷酸盐不同，基于CYS的催化机制允许发展底物捕获突变体，因此不能修改PP2C-酶的金属基催化机制以产生底物捕获突变体。此外，自然化合物可在PPP家族磷酸盐（即冈田酸和钙钙蛋白酶A）上作用，不会影响PP2C活性。因此，研究界特别需要研究PP2C家庭磷酸盐的工具。在开发了产生大量催化活性的PP2C4（已知癌基因）和荧光测定方面的方法以可靠地测量PP2C4活性时，该应用的目的是开发可用于识别该生物学重要磷酸化酶的特定或高度选择性的，高度选择性的，高度选择性的抑制剂。这项工作产生的化合物将充当强大的小分子探针，这些探针将极大地帮助阐明PP2C4在正常生物学和人类疾病中的作用。它们还可以用作开发用于人类癌症医学管理的新药的铅化合物。 公共卫生相关性：丝氨酸/苏氨酸蛋白磷酸酶2C Delta（PP2C4）已成为p53信号网络的调节剂，并且似乎充当癌基因。在开发了产生大量（PP2C4）和测量测定的方法（PP2C4）活性的方法之后，我们的建议是通过开发可以与分子文库探测生产中心网络（MLPCN）结合使用的方法来识别一种特定或高度选择性的抑制剂。这项工作产生的化合物将作为探测与（PP2C4）相关的生物学和病理学的强大工具。