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

描述（由申请人提供）：人类细胞不断暴露于各种激素、生长因子和其他影响细胞生长的药物中。为了整合和解释这些外部刺激，复杂的信号网络已经进化，它允许不同类型的细胞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细胞内信号网络，这些信号网络控制细胞生长、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，磷酸化反应是由一个大家族的蛋白激酶催化的。迄今为止，已经发现了许多作为“关键”蛋白激酶的有效和高选择性抑制剂的化合物，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PP2C亚家族磷酸盐相关的生物学作用和病理学尚不清楚。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，基于cys的催化机制允许底物捕获突变体的发展，而基于金属的pp2c酶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ppc家族磷酸盐的天然化合物（即冈田酸和花青素A）不影响PP2C活性。因此，研究人员特别需要研究pp2c家族磷酸盐的工具。已经开发出生产大量具有催化活性的PP2C4（一种已知的致癌基因）的方法和可靠测量PP2C4活性的荧光测定方法，本应用程序的目标是开发一种高通量的现成测定方法，可用于鉴定这种生物学上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，将极大地帮助阐明PP2C4在正常生物学和人类疾病中的作用。它们还可以作为先导化合物，用于开发用于人类癌症医疗管理的新药。

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).