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

描述（由申请人提供）：我的研究将集中于发现具有活性基团的新型药物样Pin1抑制剂，这些活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-脯氨酸异构酶Pin1是一种含半胱氨酸的酶，它能特异性识别脯氨酸残基附近磷酸化的丝氨酸或苏氨酸，并催化酰胺键的旋转。该蛋白在物种间高度保守，是细胞周期中有丝分裂的关键调节酶。这种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性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。第一种方法将利用已知的抑制剂来发现半胱氨酸反应性拟肽。第二个将使用一种称为基于片段的药物发现策略来识别非反应性先导物，这些先导物可以开发成半胱氨酸反应性抑制剂。反应性基团的选择将使相关的例子可以在批准的药物中找到，以确保其潜在的生物相容性。为了检测相互作用，我将使用两种方法；一种是荧光各向异性测定，使用荧光标记的肽作为已知的配体。第二个项目将使用表面等离子体共振（一种测量表面质量变化的技术）来观察固定在表面的小分子与蛋白质之间的关系。有希望的结合点的结合方向将由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阐述，以发现一种有效和选择性的半胱氨酸反应性抑制剂。最后，我将使用我发现的抑制剂和高分辨率和荧光显微镜观察染色体和细胞的其他结构元件来研究Pini在有丝分裂中的作用。有方法可以在有丝分裂的特定阶段阻止细胞，从而允许逐步分析Pin1活性。比较Pin1抑制和正常细胞的表型将为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作为癌症治疗靶点的有效性，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。Pini在有丝分裂中的研究将有助于更好地理解酶的调节功能，这一重要的细胞周期阶段，以及Pin1活性如何促进正常和疾病表型。

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.