Designed Inhibitors of Pin1 in Mitosis

描述（由申请人提供）：Pin1是一种催化磷酸丝氨酸/苏氨酸-脯氨酸（pSer/Thr-Pro）酰胺异构化的酶。Pin1被假设通过构象开关调节导致有丝分裂的信号转导，异构化细胞周期调节蛋白中的特定脯氨酸酰胺。Pin1也被证明可以调节阿尔茨海默病相关tau蛋白的活性。Pin1在调节生物过程中独特的肽基脯氨酸异构酶（PPIase）酶活性是令人感兴趣的根本原因。Pin1由两个结构域组成，它们都以不同的选择性结合pSer/Thr-Pro基序。将分别为每个结构域开发特异性配体，作为研究Pin1调控的工具。在第一个特定目标中，我们提出了针对Pin1 WW结构域的构象约束的反式脯氨酸模拟文库的设计，平行合成和检测。平行合成将使用固相连接，通过不变的磷酸盐和酰胺键偶联，以产生足够纯度的化合物进行筛选。将开发一种高通量竞争性酶联免疫吸附试验（ELISA）来检测WW结构域结合。WW结构域配体也将在催化分析中进行非竞争性抑制测试，因为我们已经证明了与WW结构域结合的肽对催化的抑制作用。在第二个目标中，文库将从我们已经合成的靶向催化结构域的化合物中衍生出来：pSer-c/s-Pro异构体、还原酰胺、酮和α -酮酰胺基序。催化结构域抑制剂将在高通量酶分析中筛选，并确定最佳抑制剂的抑制模式。在第三个目标中，将两种类型的Pint配体组合成二价抑制剂，研究Pin1的两个结构域之间的结构和动态相互作用。第四个目标是开发磷酸盐前药，以提高膜的通透性。我们还将开发对磷酸酶稳定的二氟膦酸盐。这两种修饰将允许这些化合物在整个细胞和体内使用。这些最佳配体将有助于阐明Pin1内部以及Pin1与其配体之间的结构、功能和动态关系。

DESCRIPTION (provided by applicant): Pin1 is an enzyme that catalyzes isomerization of phosphoserine/threonine-proline (pSer/Thr-Pro) amides. Pin1 is hypothesized to regulate signal transduction leading to mitosis by a conformational switch, isomerizing specific prolyl amides in cell cycle regulatory proteins. Pin1 has also been shown to regulate the activity of Alzheimer's associated tau protein. The unique peptidyl-prolyl isomerase (PPIase) enzymatic activity of Pin1 in the regulation of biological processes is interesting for fundamental reasons. Pin1 consists of two domains that both bind pSer/Thr-Pro motifs with distinct selectivity's. Specific ligands will be developed for each domain separately to be used as tools to study regulation by Pin1. In the first Specific Aim, we propose the design, parallel synthesis and assay of libraries of conformationally constrained trans- proline mimics targeted to the Pin1 WW domain. Parallel synthesis will use solid phase attachment through the invariant phosphate, and amide bond couplings to produce compounds of sufficient purity for screening. A high-throughput competitive enzyme-linked immunosorbent assay (ELISA) will be developed to assay WW domain binding. The WW domain ligands will also be tested for noncompetitive inhibition in the catalytic assay because we have shown inhibition of catalysis by a peptide binding to the WW domain. In the second Aim, libraries will be derived from compounds targeted to the catalytic domain that we have already synthesized: pSer-c/s-Pro isostere, reduced amide, ketone and alpha-ketoamide motifs. The catalytic domain inhibitors will be screened in a high-throughput enzymatic assay, and the mode of inhibition will be determined for the best inhibitors. In the third Aim, the two types of ligands for Pint will be combined into bivalent inhibitors to study the structural and dynamic interactions between the two domains of Pin1. The fourth Aim concerns development of phosphate prodrugs, masked to improve membrane permeability. We will also develop diflurophosphonates stabilized towards phosphatases. These two modifications will allow these compounds to be used in whole cells and in vivo. The best ligands will be useful in collaborations designed to elucidate the structure, function and dynamic relationships within Pin1 and between Pin1 and its ligands.