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A Generalised Approach to Derive Functionally Active Peptide Inhibitors of Transcription Factor Activity

衍生转录因子活性的功能活性肽抑制剂的通用方法

基本信息

批准号：
BB/R017956/1
负责人：
Jody Mason
金额：
$ 49.45万
依托单位：
University of Bath
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2018
资助国家：
英国
起止时间：
2018 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FR017956%2F1
关键词：
Generalised Approach Derive Functionally Active

项目摘要

We will develop and test a new intracellular peptide-library screening assay that we have created to derive functional antagonists for a family of transcription factors (bZIP proteins) implicated in disease. Using a cancer causing member that binds DNA, Activator Protein-1 (AP-1), as an exemplar we have recently established a proof-of-principle for our approach. AP-1 is a major player in cancer that functions by binding specific DNA sites to control the expression of genes involved in cellular processes such as cell growth. A major strength of our screening technique is that it selects inhibitors by their ability to bind AP-1, but also ensures they shut down its function. This ability to distinguish between AP-1 binders and those that are capable of shutting down AP-1 function is unique and addresses a problem that has hampered the search for 'functionally active' inhibitors.Since the assay is undertaken entirely inside living bacterial cells, it allows for additional benefits such as removal of library members that do not bind specifically to AP-1, as well as those that are unstable, insoluble, or degraded by enzymes. The project will generate understanding about how AP-1 binds to DNA and how its activity can be prevented, as well as creating peptides with excellent potential to be further developed into druggable molecules. We will test the potency of our peptides and peptide-derived molecules using a range of biophysical, structural, and cell-based experiments, including high-resolution imaging techniques that will allow us to study how our inhibitors work by looking at individual molecules. These experiments will shed light on how our inhibitors work looking for their ability not only to bind to AP-1 but importantly to shut down its function, we will gain an understanding of dosages required, where the inhibitors bind and how quickly, if they are stable in biological fluids, can cross biological membranes, and how they behave in cancer cell cultures where AP-1 is known to play a major role. The importance of these experiments is that we can derive a rule set for the design of inhibitors, enabling us to enhance certain properties of the inhibitors at will. In addition, this rule set can then be applied to rationally design inhibitors for this and other transcription factors.

我们将开发和测试一种新的细胞内肽文库筛选试验，该试验旨在为与疾病有关的一系列转录因子(bZIP蛋白)衍生功能拮抗剂。使用一种与DNA结合的致癌分子，激活蛋白-1(AP-1)作为范例，我们最近为我们的方法建立了一个原则证明。AP-1是癌症的主要参与者，它通过结合特定的DNA位点来控制细胞生长等细胞过程中涉及的基因的表达。我们筛选技术的一个主要优势是，它根据抑制剂与AP-1结合的能力来选择抑制剂，但也确保它们关闭其功能。这种区分AP-1结合体和那些能够关闭AP-1功能的结合体的能力是独一无二的，解决了阻碍寻找功能活性抑制剂的问题。由于检测完全在活的细菌细胞内进行，它允许其他好处，如去除不特定与AP-1结合的文库成员，以及那些不稳定、不能溶解或被酶降解的文库成员。该项目将使人们了解AP-1如何与DNA结合，如何防止其活性，以及创造出具有进一步开发成可药物分子的极佳潜力的多肽。我们将使用一系列生物物理、结构和基于细胞的实验来测试我们的多肽和多肽衍生分子的效力，包括高分辨率成像技术，这将使我们能够通过观察单个分子来研究我们的抑制剂是如何工作的。这些实验将揭示我们的抑制剂是如何工作的，寻找它们不仅与AP-1结合的能力，而且重要的是关闭其功能，我们将了解所需的剂量，抑制剂在哪里结合，如果它们在生物液体中稳定，可以多快地穿过生物膜，以及它们在已知AP-1发挥主要作用的癌细胞培养中的表现。这些实验的重要性在于，我们可以推导出抑制剂设计的规则集，使我们能够随意增强抑制剂的某些性质。此外，该规则集还可用于合理设计该转录因子和其他转录因子的抑制剂。