A Generalised Approach to Derive Functionally Active Peptide Inhibitors of Transcription Factor Activity
Lead Research Organisation:
University of Kent
Department Name: Sch of Biosciences
Abstract
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Technical Summary
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.
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.
Planned Impact
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.
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.
Publications
Brennan A
(2022)
The effect of helix-inducing constraints and downsizing upon a transcription block survival-derived functional cJun antagonist.
in Cell reports. Physical science
Brennan A
(2022)
An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.
in JACS Au
Brennan A
(2020)
Selective antagonism of cJun for cancer therapy.
in Journal of experimental & clinical cancer research : CR
Leech JT
(2022)
In vitro single molecule and bulk phase studies reveal the AP-1 transcription factor cFos binds to DNA without its partner cJun.
in The Journal of biological chemistry
Description | 1. We have discovered that cFos binds to and diffuses on dsDNA 2. We have revealed how cFos, cFos:cJun and cJun:cJun locate their target sites on DNA 3. We have developed new analytical tools that we have shared on Github for the scientific community to use 4. We have developed a new inhibitor for the cFos:cJun interaction |
Exploitation Route | At present the award is still in place, but we forsee that the outcomes may be taken forward by drug researchers and anti-cancer specialists. |
Sectors | Healthcare Pharmaceuticals and Medical Biotechnology |
Description | Our findings have been used to begin to develop inhibitors of transcription factor action. This is a substantial step forwards that could be used to engage healthcare technologists in the future. We have also identified the mechanism by which the transcription factors investigated in this proposal locate their binding sites, thereby enabling other researchers to develop new inhibitors based upon this new knowledge. These together forge the foundations for improving the quality of life for patients with diseases affected by these proteins. |
First Year Of Impact | 2021 |
Sector | Healthcare,Pharmaceuticals and Medical Biotechnology |
Impact Types | Societal |
Description | Studying the mechanism of inhibition with Jody Mason |
Organisation | University of Bath |
Department | Department of Biology and Biochemistry |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | We have begun working with the Mason group to study the mechanism of inhibition of transcription factors. This was completely facilitated by this award. Our contribution is to provide imaging expertise. |
Collaborator Contribution | The Mason lab has provided expertise and reagents for the study. |
Impact | At this early stage of the award the outcomes are yet to emerge |
Start Year | 2018 |
Title | DIMERIZATION ASSAY |
Description | Disclosed are methods, kits and cells for screening an inhibitor of association between candidate binding partners, such as for screening antagonists of amyloid peptides. The methods, kits and cells employ a reporter expression cassette and hybrid proteins. The reporter expression cassette encodes a reporter and comprises at least one DNA binding site. Each hybrid protein comprises a candidate binding partner and a component of a DNA binding protein and, upon association, form a DNA-binding complex capable of binding to the at least one binding site and inhibiting expression of the reporter. The methods, kits and cells find application, for example, in the identification of inhibitors that may be useful in treating diseases associated with protein aggregation, such as Alzheimer's Disease and Parkinson's Disease. |
IP Reference | US2023146038 |
Protection | Patent / Patent application |
Year Protection Granted | 2023 |
Licensed | Yes |
Impact | The patent has been licensed to a new spin out company. |
Title | SCREENING ASSAY |
Description | Disclosed are methods, kits and cells for screening for test compounds that are capable of inhibiting DNA-binding activity of a DNA-binding protein. The disclosed methods, kits and cells may include a reporter expression cassette that encodes a reporter expression product, wherein the reporter expression cassette comprises at least one binding site for the DNA-binding protein such that binding of the DNA-binding protein to the binding site inhibits expression of the reporter expression product. Also disclosed are methods for producing a helix-constrained peptide that may be used in the screening methods disclosed herein. The methods, kits and cells find application, for example, in the identification of antagonists that may be useful in the treatment of cancers involving the DNA-binding protein. |
IP Reference | US2021396736 |
Protection | Patent / Patent application |
Year Protection Granted | 2021 |
Licensed | Yes |
Impact | The patent has been licensed to a new spin out company. |