Discovery of cysteine-targeted inhibitors of Pin 1 for mitotic cell-based studies

发现用于基于有丝分裂细胞的研究的 Pin 1 半胱氨酸靶向抑制剂

基本信息

批准号：
7901660
负责人：
Jesse Mischa McFarland
金额：
$ 5.05万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-08-01 至 2012-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7901660
关键词：
Active Sites Amides Amino Acids Anaphase Arts Binding Binding Sites Biological Biological Assay Cancer cell line Cell Cycle Cell Cycle Stage Cell Proliferation Cells Chromosomes Complex Crystallization Crystallography Cysteine Cytokinesis Elements Ensure Enzymes Fluorescence Anisotropy Fluorescence Microscopy Fluorescence Polarization Housing Human Hydrogen Bonding Immunofluorescence Microscopy Isomerase Label Lead Libraries Ligands Link Malignant Neoplasms Measures Metaphase Methods Mitosis Mitotic Normal Cell Nuclear Magnetic Resonance Peptides Peptidylprolyl Isomerase Pharmaceutical Preparations Pharmacologic Substance Phenotype Pinus (genus)Pipecolic Acids Positioning Attribute Proline Prostate Proteins Research Resolution Role Rotation Serine Specificity Sulfhydryl Compounds Surface Surface Plasmon Resonance Techniques Threonine Time Validation analog base cancer cell cancer therapy cancer type cell growth regulation chemical synthesis covalent bond design disease phenotype drug discovery enzyme activity inhibitor/antagonist malignant breast neoplasm member mutant novel peptidomimetics piperidine public health relevance research study small molecule small molecule libraries

项目摘要

DESCRIPTION (provided by applicant): My research will focus on the discovery of novel drug-like inhibitors of Pin1 with reactive groups that can form a covalent, yet reversible bond with the amino acid cysteine in proteins. The peptidyl-prolyl isomerase Pin1 is a cysteine-containing enzyme that specifically recognizes phosphorylated serine or threonine next to proline residues and catalyzes the rotation of the amide bond. The protein is highly conserved across species and is a key regulatory enzyme of mitosis in the cell cycle. Abnormal activity of the enzyme has been strongly linked to many types of cancer. One cysteine residue in the protein is located adjacent to the active site and thus is a promising target for a new cysteine-reactive drug. I will use a two-pronged approach to discover a new inhibitor. The first will capitalize on a known inhibitor to discover a cysteine-reactive peptidomimetic. The second will use a strategy known as fragment-based drug discovery to identify non-reactive leads that can be developed into cysteine-reactive inhibitors. The reactive groups will be selected such that related examples can be found in approved pharmaceuticals to ensure their potential biological compatibility. To detect the interactions, I will use two methods; one will be a fluorescence anisotropy assay using a fluorescently-labeled peptide as the known ligand. The second will use surface plasmon resonance, a technique that measures the change in mass at a surface, to observe the association of small molecules with a protein that has been immobilized on the surface. The binding orientation of promising hits will be determined by x-ray crystallography. Molecules that interact specifically with the binding site will be elaborated by chemical synthesis for the discovery of a potent and selective cysteine-reactive inhibitor. Finally, I will study the role of Pini in mitosis using the inhibitor I discover and high-resolution and fluorescence microscopy to observe chromosomes and other structural elements of cells. Methods are available to arrest cells at specific stages of mitosis, allowing for a step-by-step analysis of Pin1 activity. Comparing the phenotypes resulting from of Pin1 inhibition and normal cells will provide exciting new results on the function of Pin1 in cellular regulation. PUBLIC HEALTH RELEVANCE: A potent, drug-like inhibitor will aid in the validation of Pini as a target for cancer treatment, as the protein is over-expressed in many cancers, including prostate and breast cancer. The study of Pini in mitosis will lead to a better understanding of the regulatory function of the enzyme, this important cell cycle stage, and how Pin1 activity contributes to normal and disease phenotypes.

描述（由申请人提供）：我的研究将集中于发现新型药物样 Pin1 抑制剂，其反应基团可以与蛋白质中的氨基酸半胱氨酸形成共价但可逆的键。肽基脯氨酰异构酶 Pin1 是一种含半胱氨酸的酶，可特异性识别脯氨酸残基旁边的磷酸化丝氨酸或苏氨酸并催化酰胺键的旋转。该蛋白质在物种间高度保守，是细胞周期中有丝分裂的关键调节酶。该酶的异常活性与许多类型的癌症密切相关。蛋白质中的一个半胱氨酸残基位于活性位点附近，因此是一种新的半胱氨酸反应药物的有希望的靶标。我将采用双管齐下的方法来发现一种新的抑制剂。第一个将利用已知的抑制剂来发现半胱氨酸反应性肽模拟物。第二个将使用一种称为基于片段的药物发现的策略来识别可开发为半胱氨酸反应性抑制剂的非反应性先导化合物。将选择反应基团，以便在批准的药物中可以找到相关示例，以确保其潜在的生物相容性。为了检测交互作用，我将使用两种方法；一种是使用荧光标记的肽作为已知配体的荧光各向异性测定。第二个将使用表面等离子共振，一种测量表面质量变化的技术，来观察小分子与固定在表面上的蛋白质的关联。有希望的命中的结合方向将由 X 射线晶体学确定。与结合位点特异性相互作用的分子将通过化学合成来精心设计，以发现有效的选择性半胱氨酸反应抑制剂。最后，我将使用我发现的抑制剂以及高分辨率荧光显微镜来研究 Pini 在有丝分裂中的作用，以观察染色体和细胞的其他结构元素。有多种方法可以将细胞阻滞在有丝分裂的特定阶段，从而可以对 Pin1 活性进行逐步分析。比较 Pin1 抑制和正常细胞产生的表型将为 Pin1 在细胞调节中的功能提供令人兴奋的新结果。公共健康相关性：一种有效的类药物抑制剂将有助于验证 Pini 作为癌症治疗靶点的作用，因为该蛋白在许多癌症中过度表达，包括前列腺癌和乳腺癌。对 Pini 在有丝分裂中的研究将有助于更好地了解酶的调节功能、这一重要的细胞周期阶段，以及 Pin1 活性如何影响正常和疾病表型。