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控机制是脯氨酸定向磷酸化某些Ser/Thr-Pro （phos.Ser/Thr-Pro）基序。Pro独特的立体化学性质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在很长一段时间内没有得到重视。我们最近发现了一种独特的脯氨酸异构酶Pin1（基因ID， NM_006221；蛋白质ID， NP_006212），它可以催化特定phos的构象开关。一组蛋白中的Ser/Thr-Pro基序调节细胞信号。这种由pin1催化的构象调节可以对多种细胞过程中的许多关键蛋白质产生深远的影响。重要的是，Pin1失调在越来越多疾病的发展中起着关键作用，并提供了一种潜在的新治疗选择。然而，迄今为止发现的抑制Pin1功能的化学探针要么缺乏急需的特异性，要么根本无法进入细胞。我们现在已经开发了一系列强大而敏感的程序来鉴定和评估体外和体内的Pin1探针，并在我们的试点筛选中发现了有趣的hit。因此，在本次提案中，我们将与dr。Douglas Auld和Craig Thomas在NIH化学基因组学中心鉴定Pin1化学探针，其具体目的如下：(1)使用定量高通量筛选方法鉴定人类Pin1抑制剂，针对含有300,000个小分子的MLSMR。(2)在二级分析中验证hit的效力和特异性，以鉴定那些特异性抑制Pin1的化合物，而不是其他非磷酸化特异性脯氨酸异构酶。(3)通过三级细胞分析、构效关系分析、结构方法、类似物合成和药物化学原理等方法对Pin1化学探针进行表征和优化。(4)在本提案的授权期限之外，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中测试最有希望的化合物抑制Pin1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来研究生理和病理条件下pin1调节的亲定向磷酸化信号。

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.