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

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AP-1 is a major player in cancer. We have developed a novel assay that screens peptide libraries inside bacterial cells to generate inhibitors that bind AP-1 and guarantee functional loss. Those that bind AP-1 but do not shut down function will be removed from the library. Additional selection pressures mitigate against non-selection by removing insoluble, unstable, aggregate prone and non-specific members; leaving excellent scaffolds for further refinement. We provide clear evidence for proof of concept in this proposal. Lastly, using a technique known as CANDI developed by Mason, inhibitors will be derived such that they can work together without cross-talk, providing potentially synergistic combinations capable of 'mopping-up' both Jun and Fos AP-1 components.

Peptides and their mimetics will be tested using a range of biophysical (ensemble and single molecule), structural, and cell-based approaches to determine how these peptides block AP-1 function. Biophysical/structural studies will characterise how effective the peptides/mimetics bind to AP-1 and shut down function. Single molecule studies will provide a direct understanding of the mechanism of inhibition to enable the design of more effective inhibitors. These studies will be complemented by cell biology experiments to verify function in cancer cells in which AP-1 is a major player in the disease (e.g. breast, colon and lung cancers where cJun is over-expressed). For the 2-3 most effective peptides we will create and test mimetics to create compounds that can be further developed into druggable molecules. These will bring drug-like properties such as stability and bioavailability while serving as constrained secondary structural mimics. These compounds will be fed back into the biophysical and cellular studies to ascertain if the mechanism of action is preserved.

The results from this research will find application in a number of important areas. The principle users of our findings will be both the academic community and those involved in developing protein-protein inhibitors. Hence the longer term beneficiaries include the health sector, where inhibitors developed as a consequence of this research will find application.
Most proteins function through interaction with other proteins; therefore making the development of a rational approach to interfering with such interactions incredibly important, and timely. This proposal will developing an understanding of how protein interaction inhibitors work and therefore will open doors into new therapies for a variety of diseases. Our example system itself will find application in the development of anti-cancer treatments

By providing a pre-defined intracellular screen for efficacy this study has the potential to transform the approach used to derive new inhibitors. Therefore we expect that our research will lead to considerable benefits for academics attempting to generate inhibitors of protein interactions both for therapeutics and non-therapeutics. Furthermore, the acceleration of the drug design process gained by using TBS to generate novel inhibitors can serve as the starting point for therapeutics against many other disease processes involving protein-protein interactions. This has considerable potential commercial benefit and therefore would benefit both the economic and knowledge economies of the UK. To develop interactions with industry we will use with the Research Services and the Innovation & Enterprise offices of both institutions.

The proposal will support two PDRAs across two institutions. The training potential is excellent as we intend for both PDRAs to interact and work together synergistically. This will hone their team work, networking, seminar and report writing skills. The techniques that we will be applying are very broad, from cell biology through protein biophysics to single molecule microscopy and the exposure of trainee scientists to this high interdisciplinary environment will be hugely beneficial. Investment in such research will enable the UK to remain internationally competitive as a knowledge economy.

All findings will be published in high impact publications to disseminate with the wider scientific community. In addition, seminars will be hosted for scientists (invited seminars and meetings), the general public (e.g. cafe scientifique, schools days at Bath, Bath Tapsin2Science), as we have strived to do over previous years. Communications outside the university will be aided by the Communications Office at Bath and the Press Office at Kent who liaise with journalists for radio/television and other media pieces, and who also produce their own monthly publications.