Designed Inhibitors of Pin1 in Mitosis

描述（由申请人提供）：Pin 1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。Pin 1被假设为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酰酰胺。Pin 1也被证明可以调节阿尔茨海默氏症相关tau蛋白的活性。Pin 1在调节生物过程中独特的肽基脯氨酰异构酶（PPIase）酶活性是令人感兴趣的基本原因。Pin 1由两个结构域组成，这两个结构域都以不同的选择性结合pSer/Thr-Pro基序。将为每个域分别开发特定的配体，以用作研究Pin 1调控的工具。在第一个具体目标中，我们提出了针对Pin 1 WW结构域的构象受限的反式脯氨酸模拟物文库的设计、平行合成和测定。平行合成将通过不变的磷酸盐和酰胺键偶联使用固相连接，以产生足够纯度的化合物用于筛选。将开发高通量竞争性酶联免疫吸附测定（ELISA）来测定WW结构域结合。还将在催化测定中测试WW结构域配体的非竞争性抑制，因为我们已经显示了通过与WW结构域结合的肽对催化的抑制。在第二个目标中，文库将来自我们已经合成的靶向催化结构域的化合物：pSer-c/s-Pro电子等排体，还原的酰胺，酮和α-酮酰胺基序。将在高通量酶测定中筛选催化结构域抑制剂，并确定最佳抑制剂的抑制模式。在第三个目标中，将Pint的两种类型的配体组合成双价抑制剂，以研究Pin 1的两个结构域之间的结构和动力学相互作用。第四个目标涉及磷酸盐前药的开发，掩蔽以改善膜渗透性。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用于全细胞和体内。最好的配体将是有用的合作，旨在阐明结构，功能和动力学的Pin 1内的关系，Pin 1和它的配体之间。

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.