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：
8110484
负责人：
Jesse Mischa McFarland
金额：
$ 2.65万
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/8110484
关键词：
Active Sites Amides Amino Acids Anaphase 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 Health Housing Human Hydrogen Bonding Immunofluorescence Microscopy Isomerase Label Lead Libraries Ligands Link Malignant Neoplasms Malignant neoplasm of prostate 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 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 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的抑制剂，其反应性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 - 培养基异构酶PIN1是一种含半胱氨酸的酶，在脯氨酸残基旁边专门识别磷酸化的丝氨酸或苏氨酸，并催化酰胺键的旋转。该蛋白质在各种物种之间是高度保守的，是细胞周期中有丝分裂的关键调节酶。酶的异常活性与多种类型的癌症密切相关。该蛋白质中的一个半胱氨酸残留物位于活性位点附近，因此是一种新的半胱氨酸反应性药物的有希望的靶标。我将使用两管沟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，发现半胱氨酸反应性肽症。第二种将使用称为基于碎片的药物发现的策略来识别可以发展为半胱氨酸反应性抑制剂的非反应性铅。将选择反应性组，以便可以在认可的药物中找到相关的示例，以确保其潜在的生物学兼容性。为了检测相互作用，我将使用两种方法。一种将是使用荧光标记的肽作为已知配体的荧光各向异性测定。第二种将使用表面等离子体共振，该技术可以测量表面的质量变化，以观察小分子与已固定在表面上的蛋白质的缔合。有希望的命中的结合方向将通过X射线晶体学确定。与结合位点特别相互作用的分子将通过化学合成来阐述，以发现有效的和选择性的半胱氨酸反应性抑制剂。最后，我将使用I发现的抑制剂以及高分辨率和荧光显微镜研究PINI在有丝分裂中的作用，以观察细胞的染色体和其他结构元素。方法可用于在有丝分裂的特定阶段逮捕细胞，从而允许对PIN1活性进行逐步分析。比较由PIN1抑制和正常细胞引起的表型将为PIN1在细胞调节中的功能提供令人兴奋的新结果。公共卫生相关性：一种有效的药物样抑制剂将有助于验证PINI作为癌症治疗的靶标，因为该蛋白在包括前列腺癌和乳腺癌在内的许多癌症中都过表达。 PINI在有丝分裂中的研究将使人们更好地了解酶的调节功能，这个重要的细胞周期阶段以及PIN1活性如何促进正常和疾病表型。