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

描述(申请人提供)：我的研究将集中在PIN1的新型类药物抑制剂的发现上，这些药物的活性基团可以与蛋白质中的氨基酸半胱氨酸形成共价的可逆键。肽基-脯氨酰异构酶Pin1是一种含有半胱氨酸的酶，它能识别靠近脯氨酸残基的磷酸化丝氨酸或苏氨酸，并催化酰胺键的旋转。该蛋白在不同物种之间高度保守，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部位附近，因此是新的半胱氨酸反应性药物的有希望的靶点。我将使用双管齐下的方法来发现一种新的抑制剂。第一个将利用一种已知的抑制剂来发现一种半胱氨酸反应性多肽模拟物。第二个将使用一种被称为基于片段的药物发现的策略来识别可以开发成半胱氨酸反应性抑制剂的非反应性先导。将选择反应性基团，以便在经批准的药物中找到相关实例，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；一种是使用荧光标记的多肽作为已知配体的荧光各向异性分析。第二个将使用表面等离子体共振，这是一种测量表面质量变化的技术，以观察小分子与固定在表面的蛋白质的联系。有希望的HITS的结合方向将通过X射线结晶学来确定。与结合部位特异相互作用的分子将通过化学合成进行细化，以发现一种有效的、选择性的半胱氨酸反应抑制物。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其他细胞结构元素来研究PINI在有丝分裂中的作用。有方法可用来阻止细胞在有丝分裂的特定阶段，允许逐步分析Pin1的活性。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。与公共健康相关：一种有效的类药物抑制剂将有助于确认PINI作为癌症治疗的靶点，因为这种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理解该酶的调节功能，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。

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.