Specific inhibition of transcription factors with Cobalt-Schiff Base Complexes

项目摘要/摘要 近年来，金属在医学上的使用有了令人印象深刻的增长，因为 深入了解具有生物活性的金属络合物和含金属蛋白质的结构。 该项目的目标是开发一个平台，专门抑制锌指转录因子(TF)， 尤其是格利一家。Gli家族转录因子代表了刺猬(HH)途径的最后一步。已经过去了- 该通路的激活与包括基底细胞癌在内的多种肿瘤的生长有关 (BCC)和髓母细胞瘤。因此，这些因子的抑制剂是生物学和生物科学的有力研究工具。 作为一类全新的治疗剂具有潜在的用途。我们正在开发钴(III)-席夫碱 通过独特的机制抑制转录因子的络合物(Co(III)-DNA)。这些复合体针对特定的 通过结合诱骗寡核苷酸来模拟目标蛋白质的天然DNA靶点。 随后钴络合物与锌指结构域中的组氨酸的配位破坏了结构 导致不可逆的转录活性抑制。 我们实验室以前的工作表明，针对Snail TFS的Co(III)-DNA阻断了乳腺癌的EMT 细胞系。我们实验室的初步研究表明，Co(III)-DNA选择性地抑制Ci(果蝇 Gli的同源物)在非哺乳动物胚胎发育模型中。在这里，新的Co(III)-DNA结合物 靶向Gli转录因子的能力将被评估为具体抑制Gli转录活性和 以哺乳动物细胞为基础的报告分析以及与疾病相关的BCC细胞系中的HH途径。在目标1中，我们 将详细研究Co(III)-Schiff碱和Co(III)-DNA与Gli DNA结合域的相互作用 氢-氚交换-电喷雾质谱联用。在目标2中，我们将检查 CO(III)-Gli DNA，比较靶向Gli和可以结合相同DNA的不同转录因子(Zic) 序列，但亲和力大大降低。在目标3中，将使用金纳米颗粒局部递送系统来 在全细胞研究和3D实验中评估靶向Gli TFS的Co(III)-DNA干扰HH途径的能力 器官型木筏培养为表皮模型。 我们在西北大学组建了一个团队，其中包括我的研究团队，该团队已经 大量Co(III)-Schiff碱二元配合物的物理和生物学性质研究 几十年。罗伯特·霍姆格伦教授是一位发展生物学家，他的研究重点是信号 转导通路及其在构图中的作用。艾米·帕勒教授，医学博士和贝瑟尼·佩雷斯-怀特教授，博士， 其研究重点是皮肤细胞的交流和发育。最后，我们增加了教授 安东尼·奥罗(斯坦福大学)是Hedgehog信号通路和罗勒细胞癌作为 顾问(见LOS)。这项研究的成功完成将产生转铁蛋白的抑制剂，其功能是 新的研究工具和一些依赖转铁蛋白信号的疾病的治疗。

Project Summary/Abstract The use of metals in medicine has grown impressively in recent years as the result of greatly advanced understanding of the structures of biologically active metal complexes and metal-containing proteins. The goal of this project is to develop a platform that specifically inhibits zinc finger transcription factors (TFs), specifically the Gli family. Gli family TFs represent the final step in the hedgehog (Hh) pathway. The over- activation of this pathway is associated with the growth of a variety of tumors including basal cell carcinoma (BCC) and medulloblastoma. Therefore, inhibitors of these TFs represent potent research tools for biology and have potential use as an entirely new class of therapeutic agents. We are developing cobalt(III)-Schiff base complexes (Co(III)-DNA) that inhibit TFs by a unique mechanism. The complexes are targeted to specific proteins by conjugating decoy oligonucleotides mimicking the native DNA target site of the protein of interest. Subsequent coordination of the cobalt complex to histidines in the zinc finger domain disrupts the structure leading to irreversible inhibition of transcriptional activity. Previous work from our lab has shown that Co(III)-DNA targeted to Snail TFs block EMT in breast cancer cell lines. Preliminary studies in our lab has shown that Co(III)-DNA selectively inhibits Ci (the Drosophila homologue of Gli) in non-mammalian embryo models of development. Here, new Co(III)-DNA conjugates targeted to Gli TFs will be evaluated for their capacity to specifically inhibit Gli transcriptional activity and the Hh pathway in mammalian cell-based reporter assays, and in disease-relevant BCC cell lines. In Aim 1, we will study the interaction of Co(III)-Schiff base and Co(III)-DNA with the Gli DNA binding domain in detail using hydrogen-deuterium exchange coupled with ESI-mass spectrometry. In Aim 2 we will examine the specificity of Co(III)-Gli DNA, comparing targeting to Gli vs. a different transcription factor (Zic) that can bind the same DNA sequence, but with much reduced affinity. In Aim 3, a gold nanoparticle topical delivery system will be used to evaluate the ability of Co(III)-DNA targeted to Gli TFs to disrupt the Hh pathway in whole cell studies and 3D organotypic raft cultures as an epidermal model. We have assembled a team at Northwestern University that includes my research group which has been investigating the physical and biological properties of large number of Co(III)-Schiff base complexes for two decades. Professor Robert Holmgren is a developmental biologist whose research focuses on signal transduction pathways and their role in patterning. Professors Amy Paller, MD and Bethany Perez-White, PhD, whose research focuses on skin cell communication and development. Finally, we have added Professor Anthony Oro (Stanford) who is world leader in Hedgehog signaling pathways and basil cell carcinoma as a consultant (see LOS). The successful completion of this research will produce inhibitors of TFs that function as new research tools and the treatment of a number of diseases that rely on TF signaling.