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

蛋白磷酸酶PP2C包含12个人类酶家族，其相关序列折叠成独特的3D结构，表现出金属离子依赖性，催化机制和底物特异性，不同于其他类型的蛋白丝氨酸/苏氨酸磷酸酶。PP2C从根本上是重要的，从酵母到人类的多个基因都是保守的，基因敲除表型表明PP2C在控制辐射损伤和其他形式的细胞应激反应中起着重要作用。PP2C异构体Wip1（野生型p53诱导磷酸酶，或PP2C或PPM1D）的转录由肿瘤抑制因子p53控制。该基因的缺失导致DNA损伤反应途径的激活，并抑制癌基因的肿瘤形成。迄今为止，还没有有效的合成或天然产物小分子抑制剂或激活剂用于任何PP2C，这阻碍了对其生物学功能的研究，并阻碍了针对该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家族的三种不同成员并行开发hts兼容的检测方法。计划生产重组、纯化的磷酸酶PP2C、PP2C和PP2C (Wip1)，并利用显色和磷酸肽底物建立HTS测定的最佳条件。该方法建立了选择性和同工异构体特异性的二次筛选，具有鉴定广谱和高度特异性的PP2C活性化学调节剂的潜力。第二个目标是开发和优化新的In-Cell Western HTS检测，通过双波长红外检测定量两种不同抗体的同时染色。这种活细胞试验将检测影响PP2C对内源性底物活性的细胞渗透性化合物。小分子修饰剂的可用性将是一项重大进展，将有利于研究不同PP2C的生物学功能，并为开发具有药用特性的新型化合物铺平道路。人体生理受体内激素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对这些信号作出反应，这些网络通过在数千种不同蛋白质上添加和去除磷酸基团来动态地开启/关闭各种过程。人类疾病是由这些激酶和磷酸酶之间的不平衡引起的，临床使用的新靶向疗法是阻断这些酶活性的化学物质。一种叫做PP2C的磷酸酶家族调节着对DNA损伤和细胞应激的反应。该项目旨在为PP2C提供特定和可靠的检测方法，以筛选数千种化学物质，并确定新的激活剂或抑制剂。PP2C的化学修饰剂具有潜在的医学应用价值，将被用作研究正常细胞和癌细胞信号传导的研究工具。

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.