Identification of Pin1 Chemical Probes for Studying Phosphorylation Signaling

描述(由申请人提供)：细胞中一种常见的中央调控机制是某些丝氨酸/苏氨酸-Pro(磷酸-丝氨酸/苏氨酸-Pro)基序上的脯氨酸指导的磷酸化。Pro独特的立体化学意味着它可以采用两种不同的顺式或反式构象，但这些构象开关的生物学意义长期以来没有得到重视。我们最近发现了一种独特的脯氨酰异构酶，PIN1(基因ID，NM_006221；蛋白质ID，NP_006212)，它催化特定磷酸的构象开关。丝氨酸/苏氨酸-Pro基序在一组蛋白质中调节细胞信号。这种由Pin1催化的构象调节可以在不同的细胞过程中对许多关键蛋白质产生深远的影响。重要的是，Pin1的解除调控在越来越多的疾病的发展中起着关键作用，并提供了一种潜在的新的治疗选择。然而，到目前为止发现的抑制Pin1功能的化学探针要么缺乏急需的特异性，要么根本不能进入细胞。我们现在已经开发了一系列强大和灵敏的程序来在体外和体内识别和评估Pin1探针，并在我们的试点屏幕上识别了有趣的命中。因此，在本提案中，我们将与NIH化学基因组中心的Douglas Auld和Craig Thomas博士合作，寻找Pin1化学探针，具体目标如下：(1)针对包含300,000个小分子的MLSMR，使用定量高通量筛选方法来识别人Pin1的抑制剂。(2)在二次实验中验证HITS的效力和特异性，以确定那些特异性地抑制Pin1的化合物，而不是其他非磷酸化特异性的Pro-L异构酶。(3)通过三级细胞分析、构效关系分析、构效关系分析、类似物合成和药用化学原理等方法对Pin1化学探针进行表征和优化。(4)在这项建议的授权期之后，在我们实验室建立的几种与Pin1相关的癌症或阿尔茨海默病小鼠模型上，测试最有希望的化合物抑制Pin1功能的能力。这一建议将使我们能够找到迫切需要的化学探针来研究生理和病理条件下Pin1调节的前向磷酸化信号。 与公共健康相关：我们最近发现了一种名为Pin1的新酶，它是许多细胞过程的关键调节因子。重要的是，Pin1基因的解除在越来越多的人类疾病的发展过程中发挥了关键作用，这些疾病包括衰老、癌症和阿尔茨海默病和S病。然而，目前还没有Pin1特异的化学探针可用。在这一应用中，我们建议发现并优化Pin1化学探针，以研究Pin1在生理和病理条件下的调控过程。

DESCRIPTION (provided by applicant): A common and central regulatory mechanism in the cell is proline-directed phosphorylation on certain Ser/Thr- Pro (phos.Ser/Thr-Pro) motifs. The unique stereochemistry of Pro means that it can adopt two distinct cis or trans conformations, but the biological significance of these conformational switches was unappreciated for a long time. We have recently identified a unique prolyl isomerase, Pin1 (Gene ID, NM_006221; Protein ID, NP_006212) that catalyzes the conformational switches of specific phos.Ser/Thr-Pro motifs in a subset of proteins to regulate cell signaling. Such Pin1-catalyzed conformational regulation can have a profound impact on many key proteins in diverse cellular processes. Importantly, Pin1 deregulation plays a pivotal role in the development of an increasing number of diseases and provides a potential new therapeutic option. However, chemical probes to inhibit Pin1 function identified so far either lack the critically needed specificity or simply cannot enter cells. We have now developed a series of robust and sensitive procedures to identify and evaluate Pin1 probes in vitro and in vivo and also identified interesting hits in our pilot screen. Therefore, in this proposal, we will collaborate with Drs. Douglas Auld and Craig Thomas at NIH Chemical Genomics Center to identify Pin1 chemical probes, with the following specific aims: (1) To identify inhibitors of human Pin1 using a quantitative high-throughput screening approach against the MLSMR containing 300,000 small molecules. (2) To validate the potency and specificity of the hits in secondary assays to identify those compounds that specifically inhibit Pin1, but not other non-phosphorylation-specific prolyl isomerases. (3) To characterize and optimize Pin1 chemical probes by tertiary cell-based assays and structure-activity relationship analysis, structure-based methods, analogue synthesis and medicinal chemical principles. (4) Beyond the granting period for this proposal, to test the most promising compounds for their ability to inhibit Pin1 function in several Pin1-relevant mouse models of cancer or Alzheimer<s disease that we have established in our laboratory. This proposal would allow us to identify urgently needed chemical probes to study Pin1-regulated Pro-directed phosphorylation signaling under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: We have recently identified a new enzyme called Pin1 that is a pivotal regulator of numerous cellular processes. Importantly, Pin1 deregulation plays a critical role in the development of an increasing number of human diseases, including aging, cancer and Alzheimer<s disease. However, currently there is no Pin1-specific chemical probe available. In this application, we propose to discover and optimize Pin1 chemical probe to study Pin1-regulated processes under physiological and pathological conditions.