THE ROLE OF PIN1 IN MITOSIS WITH DESIGNED INHIBITORS

描述：PIN1，一种新发现的有丝分裂信号调节因子 转导，是一种作用于磷酸丝氨酸-脯氨酸酰胺的酶。这个 PIN1独特的磷酸化依赖的肽基-Pro异构酶活性 调节有丝分裂使其成为潜在癌症的极具吸引力的新靶点 化疗药物。Pin 1被认为是通过一种 构象转换，使一系列蛋白质中的关键脯氨酰胺异构化， 在有丝分裂过程中上调。CDC25特别是已知与 Pin1通过两个磷酸丝氨酸-脯氨酸基序。CDC25磷酸酶调节 有丝分裂特异的激酶CDC2与细胞周期蛋白B络合的活性 Pro需要将Pro之前的酰胺键异构化 反式和顺式构象以便折叠成具有生物活性的 构象。肽基-脯氨基加速了折叠过程 异构酶(PPIase酶)。更好地机械地理解原住民 Pin 1酶的PPIase活性将有助于了解PPIase的依赖 流程。第一个具体目标涉及通过构象抑制Pin1 受约束的顺式和反式脯氨酸底物模拟。模仿将会是 通过立体选择性有机合成技术合成的 派的实验室。(Z)和(E)-烯基脯氨酸二肽模拟物不能 异构化，因此它们将被检测为Pin1的竞争性抑制剂。这个 CDC25的顺式和反式脯氨酸模拟物对Pin1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……里面 第二个具体目标是，激酶上游的构象选择性 磷酸化CDC25的Pin1将被研究。Pin 1在人类免疫系统中的作用 有丝分裂将被研究：1)作为一种磷酸Ser-Pro异构酶 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)在CDC25蛋白折叠和 陪护研究。在第三个具体目标中，Pin1的作用机制 将利用同位素效应研究肽基-脯氨酸异构酶的活性。 活性中心硫醇滴定。利用这些信息，一系列 将合成基于机理的抑制剂，并检测其对Pin1的抑制作用。 基于机理的抑制剂被认为是合理的设计线索 抗癌药物。这项提案的成功完成将导致 了解Pin1 PPIase的活性机制有助于阐明其 在导致有丝分裂的信号转导中起重要作用。

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.