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

我们将开发和测试一种新的细胞内肽库筛选试验，我们已经创建了衍生功能性拮抗剂家族的转录因子（bZIP蛋白）参与疾病。使用结合DNA的致癌成员激活蛋白-1（AP-1）作为范例，我们最近为我们的方法建立了原理证明。AP-1是癌症中的主要参与者，通过结合特定的DNA位点来控制参与细胞过程（如细胞生长）的基因表达。我们的筛选技术的一个主要优势是，它通过抑制剂结合AP-1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结合剂和那些能够关闭AP-1功能的结合剂的能力是独特的，并且解决了阻碍寻找“功能活性”抑制剂的问题。由于该测定完全在活细菌细胞内进行，它允许额外的益处，例如去除不特异性结合AP-1的文库成员，以及那些不稳定、不溶性、或被酶降解。该项目将产生关于AP-1如何与DNA结合以及如何防止其活性的理解，以及创造具有进一步开发成可药用分子的良好潜力的肽。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肽和肽衍生分子的效力，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如何工作。这些实验将阐明我们的抑制剂如何工作，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，抑制剂结合的位置以及如果它们在生物液体中稳定，可以穿过生物膜的速度，以及它们在AP-1已知发挥主要作用的癌细胞培养物中的行为。这些实验的重要性在于，我们可以推导出一套设计抑制剂的规则，使我们能够随意增强抑制剂的某些特性。此外，该规则集可以应用于合理设计这种和其他转录因子的抑制剂。

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.