THE ROLE OF PIN1 IN MITOSIS WITH DESIGNED INHIBITORS

PIN1 在有丝分裂中与设计抑制剂的作用

• 批准号: 6387319
• 负责人: FELICIA A ETZKORN
• 金额: $ 17.87万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387319
• 关键词: biological signal transduction; cell cycle; cell cycle proteins; cell growth regulation; conformation; drug design /synthesis /production; enzyme activity; enzyme inhibitors; enzyme mechanism; enzyme substrate analog; molecular chaperones; peptidylprolyl isomerase; phosphoprotein phosphatase; phosphoserine; proline; protein folding

DESCRIPTION: Pin 1, a newly discovered regulator of mitosis signal transduction, is an enzyme that acts on phosphoserine-proline amides. The unique phosphorylation dependent peptidyl-prolyl isomerase activity of Pin1 in regulating mitosis makes it a very attractive novel target for potential cancer chemotherapeutics. Pin 1 is hypothesized to regulate mitosis by a conformational switch, isomerizing key proline amides in a set of proteins that are upregulated during mitosis. Cdc25 specifically is known to interact with Pin1 through two phosphoserine-proline motifs. Cdc25 phosphatase regulates the activity of the mitosis-specific kinase, Cdc2 complexed with cyclin B. Many prolines require isomerization of the amide bond preceding proline between the trans and cis conformations in order to fold into the biologically active conformation. the folding process is accelerated by the peptidyl-prolyl isomerase (PPIase enzymes). A better mechanistic understanding of the native PPIase activity of the Pin 1 enzyme will give insight into PPIase-dependent processes. The first Specific Aim concerns Pin1 inhibition by conformationally constrained cis- and trans- proline substrate mimics. The mimics will be synthesized by stereoselective organic synthesis techniques developed in the PI's laboratory. The (Z) and (E)-alkene proline dipeptide mimics cannot be isomerized, so they will be assayed as competitive inhibitors of Pin1. The relative levels of Pin1 inhibition by cis and trans proline mimics of the Cdc25 substrate will help elucidate the mechanism by which Pin regulates mitosis. In the second specific aim, the conformational selectivity of kinases upstream of Pin1 that phosphorylate Cdc25 will be investigated. The role of Pin 1 in mitosis will be investigated: 1) as an enzyme that isomerases phosphoSer-Pro bonds in the substrate, Cdc25 phosphatase; 2) as a cofactor that binds to Cdc25 to regulate its activity in mitosis and 3) in Cdc25 protein folding and chaperone studies. In the third specific aim, the mechanism of Pin1 peptidyl-proline isomerase activity will be investigated using isotope effects and active site thiol titration. Using this information, a series of mechanism-based inhibitors will be synthesized and assayed for Pin1 inhibition. The mechanism-based inhibitors are considered rational design leads for anti-cancer drugs. The successful completion of this proposal will lead to an understanding of the mechanism of Pin1 PPIase activity to help elucidate its essential role in signal transduction leading to mitosis.

描述：引脚1，新发现的有丝分裂信号调节器 转导，是一种作用于磷serine--磷酰胺的酶。这 PIN1在 调节有丝分裂使其成为潜在癌症的非常吸引人的新型目标 化学治疗学。销钉1被认为通过A调节有丝分裂 构象开关，在一组蛋白质中与关键脯氨酸酰胺异构化 在有丝分裂过程中被上调。 CDC25特别已知可以与 PIN1通过两个磷酸盐丙啉基序。 CDC25磷酸酶调节 有丝分裂特异性激酶的活性，与细胞周期蛋白B复合。许多 五 反式和顺式构象以折叠到生物上活跃 构象。折叠过程被肽基蛋白酶加速 异构酶（PPIASE酶）。对本地的更好的机械理解 引脚1酶的PPIASE活性将洞悉依赖PPIASE 过程。第一个特定目的涉及构象抑制PIN1 受约束的顺式和泛脯氨酸底物模拟物。模仿将是 由立体选择性有机合成技术合成的 PI的实验室。 （z）和（e） - 烷烃脯氨酸二肽模拟物不能是 异构化，因此将它们作为PIN1的竞争抑制剂进行测定。这 顺式抑制PIN1的相对水平和Cdc25的反式脯氨酸模拟物的相对水平 底物将有助于阐明PIN调节有丝分裂的机制。在 第二个特定目的是上游激酶的构象选择性 PIN1将研究该磷酸化CDC25。引脚1在 有丝分裂将研究：1）作为异构酶磷酸化蛋白酶的酶 底物中的键Cdc25磷酸酶； 2）作为与Cdc25结合的辅因子 调节其在有丝分裂中的活性，3）Cdc25蛋白质折叠和 伴侣研究。在第三个特定目的中，PIN1的机制 将使用同位素效应研究肽基 - 丙啉异构酶活性 和主动位点硫醇滴定。使用此信息，一系列 基于机制的抑制剂将合成并测定以抑制PIN1。 基于机制的抑制剂被认为是理性的设计线索 抗癌药。该提案的成功完成将导致 了解PIN1 PPIASE活性的机制，以帮助阐明其 在信号转导导致有丝分裂的信号转导中的重要作用。