HTP screening for inhibitors of ser/the protein phosphatase 5

描述（由申请人提供）：体内几乎每个细胞都不断暴露于各种激素、生长因子和其他影响细胞生物功能的物质中。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。在真核细胞中，蛋白质的可逆磷酸化调节了许多控制细胞生长、代谢、分化、衰老和凋亡的信号网络。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测蛋白激酶的生物学和病理学作用的有力工具。相比之下，人们对蛋白磷酸酶的生物学作用知之甚少。在很大程度上，这是由于缺乏特异性或高选择性的小分子抑制剂。我们已经开发出生产大量催化活性丝氨酸/苏氨酸蛋白磷酸酶5 （PP5）的方法，一种快速可靠地测量PP5活性的荧光法，并以高分辨率（1.6 E）解决了PP5的晶体结构，我们现在有了开发PP5抑制剂的工具。本提案的目的是鉴定一种特异性或高选择性的PP5抑制剂。有两个具体目标。目的1。与分子文库探针生产中心网络（MLPCN）一起进行htp筛选，以确定抑制PP5催化活性的化合物。目标2。针对结构相关的磷酸酶，对Aim 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（例如提高效力/特异性）。由此产生的化合物将作为强大的小分子探针，极大地帮助阐明PP5所起的生物学和病理学作用。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): 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.