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1，一个新发现的有丝分裂信号调节器 转导是作用于磷酸丝氨酸-脯氨酸酰胺的酶。的 Pin 1独特的磷酸化依赖性肽基脯氨酰异构酶活性， 调节有丝分裂使其成为潜在癌症的非常有吸引力的新靶点 化疗药物Pin 1被假设为通过一个 构象开关，异构化一组蛋白质中的关键脯氨酸酰胺， 在有丝分裂过程中被上调。已知cdc 25特别与 Pin 1通过两个磷酸丝氨酸-脯氨酸基序。Cdc 25磷酸酶调节 有丝分裂特异性激酶Cdc 2与细胞周期蛋白B复合的活性。许多 脯氨酸需要在脯氨酸之前的酰胺键的异构化， 反式和顺式构象，以便折叠成生物活性的 构象肽基脯氨酰加速了折叠过程 异构酶（PPIase酶）。更好地机械地理解 Pin 1酶的PPIase活性将使人们了解PPIase依赖性 流程.第一个具体目标涉及构象对Pin 1的抑制 限制性顺式和反式脯氨酸底物模拟物。这些模仿者将 合成的立体选择性有机合成技术开发的 PI的实验室（Z）和（E）-烯烃脯氨酸二肽模拟物不能是 异构化，因此它们将被测定为Pin 1的竞争性抑制剂。的 Cdc 25的顺式和反式脯氨酸模拟物对Pin 1抑制的相对水平 底物将有助于阐明Pin调节有丝分裂的机制。在 第二个具体目标，激酶上游的构象选择性， 将研究磷酸化Cdc 25的Pin 1。Pin 1的作用 有丝分裂将被研究：1）作为一种酶，异构化磷酸丝氨酸-Pro 底物Cdc 25磷酸酶中的键; 2）作为与Cdc 25结合的辅因子 调节其在有丝分裂中的活性和3）Cdc 25蛋白折叠， 伴侣研究。在第三个具体目标中，Pin 1的机制 肽基脯氨酸异构酶活性将使用同位素效应进行研究 和活性位点硫醇滴定。利用这些信息，一系列 将合成基于机制的抑制剂并测定Pin 1抑制。 基于机理的抑制剂被认为是合理的设计， 抗癌药成功完成这一提案将导致 了解Pin 1 PPIase活性的机制，以帮助阐明其 在导致有丝分裂的信号转导中起重要作用。