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

蛋白 Ser/Thr 磷酸酶 2C 修饰物的三重威胁筛查

基本信息

批准号：
7555514
负责人：
DAVID L. BRAUTIGAN
金额：
$ 15.15万
依托单位：
UNIVERSITY OF VIRGINIA
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-06-15 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7555514
关键词：
ANXA5 gene ATM Signaling Pathway Active Sites Affect Affinity Antibodies Binding Sites Biological Assay Biological Factors Biological Process Cell Cycle Progression Cells Cellular Stress Chemical Modifier Chemicals Chromogenic Substrates Class Clinical Collection Compatible Condition Cyclin-Dependent Kinases DNA Damage Dependence Detection Development Diabetes Mellitus Disaccharides Drug Delivery Systems Environment Enzymes Excision Exhibits Family Feedback Gene Deletion Genes Genetic Transcription Grant Hormones Human Individual Inflammation Investigation Ionizing radiation Ions Laboratories Life Malignant Neoplasms Metals Okadaic Acid Oncogenes PPM1D gene Pathway interactions Phenotype Phosphopeptides Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Processes Physiology Positioning Attribute Process Property Protein Dephosphorylation Protein Isoforms Protein Kinase Protein Phosphatase 2A Regulatory Subunit PR53 Protein p53 Protein phosphatase Proteins RNA Interference Radiation Reaction Recombinants Research Resistance Role Screening procedure Signal Transduction Site Specificity Staining method Stains Stress Substrate Specificity System TP53 gene Thinking Urination Work Yeasts annexin A5 calyculin A cancer cell cell transformation cytokine enzyme activity high throughput screening human disease inhibitor/antagonist inorganic phosphate knockout gene member microcystin nitrophenylphosphate novel novel therapeutics prevent protein phosphatase 2C protein phosphatase 6 response small molecule small molecule libraries three dimensional structure tool tumor

项目摘要

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.

描述（由申请人提供）：摘要蛋白磷酸酶 PP2C 包含十几种具有相关序列的人类酶家族，这些酶折叠成独特的 3D 结构，该结构表现出与其他类型的蛋白质 Ser/Thr 不同的金属离子依赖性、催化机制和底物特异性磷酸酶。 PP2C 至关重要，从酵母到人类都有多个基因保守，基因敲除表型表明 PP2C 控制着对辐射损伤和其他形式的细胞应激的反应。称为 Wip1（野生型 p53 诱导磷酸酶；或 PP2C 或 PPM1D）的 PP2C 同种型的转录由肿瘤抑制因子 p53 控制。该基因的缺失会导致 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，还没有针对任何 PP2C 的有效合成或天然产物小分子抑制剂或激活剂，这阻碍了对其生物学功能的研究，并阻止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，针对 PP2C 家族的三个不同成员并行开发 HTS 兼容检测。该计划是生产重组、纯化的磷酸酶 PP2C、PP2C 和 PP2C (Wip1)，并建立使用显色底物和磷酸肽底物进行 HTS 测定的最佳条件。该方法建立了选择性和亚型特异性的二次筛选，并且有潜力鉴定 PP2C 活性的广谱和高度特异性的化学调节剂。第二个目标是开发和优化一种新的 In-Cell Western HTS 测定，通过双波长红外检测对两种不同抗体的同时染色进行定量。这种活细胞测定将检测影响 PP2C 针对内源性底物活性的细胞渗透性化合物。小分子修饰剂的出现将是一个重大进步，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。项目叙述人类生理学受内部激素信号和外部环境变化的控制。细胞通过蛋白激酶和磷酸酶网络响应这些信号，通过添加和去除数千种不同蛋白质上的磷酸基团来动态地打开/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。称为 PP2C 的磷酸酶家族可调节对 DNA 损伤和细胞应激的反应。该项目旨在为 PP2C 提供特定且可靠的检测方法，以筛选数千种化学物质的集合并识别新型激活剂或抑制剂。 PP2C 的化学修饰剂具有潜在的医学应用，将用作研究正常细胞和癌细胞中信号传导的研究工具。