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

概述：蛋白质磷酸酶PP2C包括一系列具有相关序列的人类酶，这些酶折叠成一个独特的3D结构，表现出与其他类型的丝氨酸/苏氨酸磷酸酶不同的金属离子依赖性、催化机制和底物专一性。PP2C是至关重要的，从酵母到人类都有多个保守的基因，基因敲除表型表明PP2C参与控制对辐射损伤和其他形式的细胞应激的反应。PP2C亚型Wip1(野生型P53诱导的磷酸酶，或PP2C或PPM1D)的转录受肿瘤抑制基因P53的控制。该基因的缺失导致DNA损伤反应通路的激活，并通过癌基因抑制肿瘤的形成。到目前为止，还没有针对任何PP2C的有效的合成或天然产品小分子抑制剂或激活剂，这阻碍了对其生物学功能的研究，并阻碍了针对这一调节酶家族的药物的开发。该项目提出了一种三重威胁方法，为PP2C家族的三个不同成员并行开发与HTS兼容的检测方法。该计划是生产重组的、纯化的磷酸酶PP2C、PP2C和PP2C(Wip1)，并建立使用发色底物和磷肽底物进行HTS分析的最佳条件。这种方法建立了选择性和异构体特异性的二次筛选，并有可能识别PP2C活性的广谱和高度特异的化学调节剂。第二个目标是开发和优化一种新的细胞内Western HTS检测方法，该方法通过双波长红外检测来定量两种不同抗体的同时染色。这项活细胞检测将检测影响PP2C对内源底物活性的细胞通透性化合物。小分子修饰剂的问世将是一项重大进展，将有助于研究不同PP2C的生物学功能，并为开发具有药用性能的新型化合物铺平道路。项目叙述人类生理学受内部荷尔蒙信号和外部环境变化的支配。细胞通过蛋白激酶和磷酸酶网络来响应这些信号，这些网络通过增加和去除数千种不同蛋白质上的磷酸基团来动态地开启/关闭各种过程。人类疾病是由这些酶和磷酸酶之间的失衡引起的，临床上使用的新的靶向治疗是阻止这些酶活性的化学物质。一种名为PP2C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PP2C产生具体和可靠的分析，以筛选数千种化学物质的集合，并确定新的激活剂或抑制剂。PP2C的化学修饰物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的信号转导的研究工具。

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.