Identification of Pin1 Chemical Probes for Studying Phosphorylation Signaling

描述（由申请人提供）：细胞中常见的中心调节机制是某些Ser/Thr-Pro（phos.Ser/Thr-Pro）基序上的脯氨酸定向磷酸化。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，但这些构象转换的生物学意义很长一段时间没有得到重视。我们最近鉴定了一种独特的脯氨酰异构酶Pin 1（Gene ID，NM_006221; Protein ID，NP_006212），其催化蛋白质亚组中特定磷酸、丝氨酸/苏氨酸-脯氨酸基序的构象转换以调节细胞信号传导。这种Pin 1催化的构象调节可以对多种细胞过程中的许多关键蛋白产生深远的影响。重要的是，Pin 1失调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，迄今为止发现的抑制Pin 1功能的化学探针要么缺乏急需的特异性，要么根本无法进入细胞。我们现在已经开发了一系列稳健和灵敏的程序来识别和评估Pin 1探针在体外和体内，也确定了有趣的命中在我们的试点屏幕。因此，在本提案中，我们将与NIH化学基因组学中心的道格拉斯奥尔德和克雷格托马斯博士合作，鉴定Pin 1化学探针，具体目标如下：（1）针对含有30万个小分子的MLSMR，使用定量高通量筛选方法鉴定人Pin 1的抑制剂。(2)验证二级试验中命中的效价和特异性，以鉴定特异性抑制Pin 1但不抑制其他非磷酸化特异性脯氨酰异构酶的化合物。(3)通过三级细胞分析和构效关系分析、基于结构的方法、类似物合成和药物化学原理对Pin 1化学探针进行表征和优化。(4)在本提案的授权期之外，测试最有希望的化合物在我们实验室建立的几种Pin 1相关的癌症或阿尔茨海默病小鼠模型中抑制Pin 1功能的能力。这一提议将使我们能够确定迫切需要的化学探针，以研究在生理和病理条件下Pin 1调节的Pro定向磷酸化信号。 公共卫生相关性：我们最近发现了一种名为Pin 1的新酶，它是许多细胞过程的关键调节因子。重要的是，Pin 1失调在越来越多的人类疾病的发展中起着关键作用，包括衰老，癌症和阿尔茨海默病。然而，目前没有可用的Pin 1特异性化学探针。在这个应用中，我们建议发现和优化Pin 1化学探针，以研究Pin 1在生理和病理条件下的调节过程。

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.