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

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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.