Designed Inhibitors of Pin1 in Mitosis

有丝分裂中 Pin1 的设计抑制剂

• 批准号: 7031406
• 负责人: FELICIA A ETZKORN
• 金额: $ 19.21万
• 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 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7031406
• 关键词: X ray crystallography; active sites; amides; biomimetics; catalyst; cell cycle; chemical bond; chemical synthesis; combinatorial chemistry; conformation; enzyme activity; enzyme inhibitors; enzyme linked immunosorbent assay; enzyme structure; high throughput technology; membrane permeability; method development; nuclear magnetic resonance spectroscopy; peptidylprolyl isomerase; phosphates; phosphonate; prodrugs; proline

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.

描述（由申请人提供）：PIN1是一种酶，可催化磷serine/苏氨酸 - 丙啉（PSER/THR-PRO）酰胺的异构化。假设PIN1可以调节信号转导，从而导致构象转换导致有丝分裂，从而使细胞周期调节蛋白中的特异性prolyl酰胺异构化。 PIN1还显示出调节阿尔茨海默氏症相关的tau蛋白的活性。在生物过程调节中，PIN1的独特肽基蛋白酶异构酶（PPIASE）酶活性很有趣。 PIN1由两个结合PSER/THR-PRO基序具有不同选择性的域组成。将为每个域分别开发特定的配体，以用作通过PIN1研究调节的工具。在第一个特定目的中，我们提出了针对PIN1 WW域的构型约束的透析模拟构型的跨性脯氨酸模拟物的设计，并行合成和测定。平行合成将通过不变磷酸盐和酰胺键耦合使用固相附着，以产生足够纯度的化合物以筛选。将开发出高通量竞争性酶联免疫吸附测定法（ELISA）来测定WW域结合。 WW结构域配体还将测试催化测定中的非竞争性抑制作用，因为我们已经显示通过肽与WW结构域结合的肽抑制催化。在第二个目标中，库将来自我们已经合成的催化域的化合物：pser-c/s-pro Isostere，降低酰胺，酮和α-酮酰胺基序。催化结构域抑制剂将在高通量酶测定中进行筛选，并将确定最佳抑制剂的抑制模式。在第三个目标中，两种类型的品脱的配体将合并为二价抑制剂，以研究PIN1的两个结构域之间的结构和动态相互作用。第四目的涉及磷酸前药的发展，掩盖以提高膜渗透性。我们还将发展为磷酸酶稳定的差异膦酸酯。这两种修饰将允许这些化合物用于整个细胞和体内。最好的配体将在旨在阐明PIN1内以及PIN1及其配体之间的结构，功能和动态关系的协作中很有用。