HTP screening for inhibitors of ser/the protein phosphatase 5

描述(申请人提供)：几乎身体中的每个细胞都不断地暴露在各种激素、生长因子和其他影响细胞生物学功能的因素中。为了整合和解释这些外部刺激，复杂的信号网络已经进化出来，允许不同类型的细胞对环境做出适当的反应。在真核细胞中，蛋白质的可逆磷酸化调节许多信号网络，这些信号网络控制着细胞的生长、代谢、分化、衰老和凋亡。蛋白质的磷酸化主要发生在丝氨酸、苏氨酸和酪氨酸残基上，而磷酸化反应是由一大类蛋白激酶催化的。到目前为止，已经发现了许多化合物，它们是“关键”蛋白激酶的有效和高选择性的抑制剂，这些抑制剂已经被证明是探索蛋白激酶的生物学和病理作用的有力工具。相比之下，人们对蛋白磷酸酶的生物学作用知之甚少。这在很大程度上是由于缺乏特异性或高度选择性的小分子抑制剂。开发了大量生产具有催化活性的丝氨酸/苏氨酸蛋白磷酸酶5(PP5)的方法，这是一种快速可靠地测量PP5活性的荧光分析方法，并在高分辨率(1.6E)下解决了PP5的晶体结构，现在我们有了开发PP5抑制剂的工具。这项建议的目的是确定一种特定的或高度选择性的PP5抑制剂。有两个具体目标。目的1.结合分子文库探针生产中心网络(MLPCN)进行HTP筛选，以确定抑制PP5催化活性的化合物。目的2.对照结构相关的磷酸酶对目标1中鉴定的化合物进行反向筛选，以确定选择性，表征抑制剂，并与MLPNC成员合作进行探针优化(例如提高效价/特异性)。通过这一努力产生的化合物将成为强大的小分子探针，将极大地帮助阐明PP5所扮演的生物和病理角色。它们还可以作为先导化合物，用于开发治疗人类癌症的新药。 与公共健康相关：我们已经确定了一种蛋白磷酸酶(PP5)，它似乎是细胞生长的关键调节因子。在开发了大量生产PP5的方法和测量PP5活性的方法后，我们建议通过与分子文库探针生产中心网络(MLPCN)一起进行大规模筛选来鉴定PP5的特异性或高选择性抑制剂。通过这一努力产生的化合物将成为强大的探针，将极大地帮助阐明PP5所发挥的生物学和病理作用。

DESCRIPTION (provided by applicant): Almost every cell in the body is constantly exposed to a variety of hormones, growth factors and other agents that influence the biological functions of cells. 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 eukaryotic cells the reversible phosphorylation of proteins regulates many signaling networks that control cell growth, metabolism, differentiation, senescence, and 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 biological and pathological actions of protein kinases. In contrast, much less is known about the biological roles of protein phosphatases. To a large extent this is due to a lack of specific or highly selective small molecule inhibitors. Having developed methods to produce large amounts of catalytically active ser/thr protein phosphatase 5 (PP5), a fluorescent assay to rapidly and reliably measure PP5 activity, and having solved the crystal structure of PP5 at high resolution (1.6 E), we now have the tools in place to develop inhibitors of PP5. The objective of this proposal is to identify a specific or highly selective inhibitor of PP5. There are two specific aims. Aim 1. Conduct a HTP-screen in conjunction with the Molecular Libraries Probe Production Centers Network (MLPCN) to identify compounds that inhibit the catalytic activity of PP5. Aim 2. Back screen compounds identified in Aim 1 against the structurally related phosphatases to determine selectivity, characterize the inhibitors, and work with members of the MLPNC for probe optimization (e.g. increase potency/specificity). The compounds produced from this effort will serve as powerful small molecule probes that will greatly aid the elucidation of the biological and pathological roles played by PP5. They may also serve as lead compounds for the development of new drugs for medical management of human cancer. PUBLIC HEALTH RELEVANCE: We have identified a protein phosphatase (PP5) that seems to be a key regulator of cell growth. Having developed methods to produce large amounts of PP5 and an assay to measure PP5 activity, we proposal is to identify a specific or highly selective inhibitor of PP5 by conducting 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 probes that will greatly aid the elucidation of the biological and pathological roles played by PP5.