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

描述（由申请人提供）：人体细胞持续暴露于各种激素、生长因子和其他影响细胞生长的试剂。为了整合和解释这些外部刺激，进化出了复杂的信号网络，使不同类型的细胞能够对其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控制细胞生长、分化、衰老和程序性细胞死亡（凋亡）的细胞内信号网络。蛋白质磷酸化主要发生在丝氨酸、苏氨酸和酪氨酸残基上，并且磷酸化反应由蛋白激酶的大家族催化。迄今为止，已经鉴定了许多化合物，其作为“关键”蛋白激酶的有效和高选择性抑制剂起作用，并且这些抑制剂已被证明是探测与蛋白激酶的作用相关的生物学和病理学的有力工具。相比之下，对蛋白磷酸盐的生物学知之甚少。值得注意的是，与属于PP 2C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。与双特异性和酪氨酸磷酸不同，其中基于Cys的催化机制允许底物捕获突变体的发展，PP 2C酶的基于金属的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（即冈田酸和calyculin A）的天然化合物不会影响PP 2C活性。因此，研究PP 2C家族磷酸盐的工具是研究界特别需要的。已经开发了产生大量催化活性PP 2C 4（已知的致癌基因）的方法和可靠地测量PP 2C 4活性的荧光测定，本申请的目的是开发高通量就绪测定，其可用于鉴定这种生物学重要的磷酸酶的特异性或高选择性抑制剂。从这项工作中产生的化合物将作为强大的小分子探针，这将大大有助于阐明PP 2C 4在正常生物学和人类疾病中的作用。它们还可以作为开发用于人类癌症医疗管理的新药的先导化合物。 公共卫生相关性：丝氨酸/苏氨酸蛋白磷酸酶2C δ（PP 2C 4）已作为p53信号传导网络的调节剂出现，并且似乎作为致癌基因起作用。在开发了产生大量（PP 2C 4）的方法和测量（PP 2C 4）活性的测定法之后，我们建议通过开发能够与分子库探针生产中心网络（MLPCN）结合进行大规模筛选的方法来鉴定特异性或高选择性抑制剂。从这项工作中产生的化合物将作为探测与（PP 2C 4）相关的生物学和病理学的有力工具。

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).