68 Projects, page 1 of 14
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.
Transducers are devices that can convert electrical energy into mechanical energy and vice versa. They are widely used in non-destructive testing to generate acoustic signals in test materials and to detect changes in the acoustic signal as it travels enabling material properties to be determined. The application areas for transducers in non-destructive testing are diverse and range from locating cracks in metal structures to diagnosing disease in humans. Transducers are typically made from single crystals such as quartz or ceramics. Recently it has been shown that a much wider range of materials can be used in transducers if they are miniaturised down to a nanometre scale. In fact, it has been shown that electrical energy can be converted to mechanical energy in biological membranes. Further, strategies to greatly increase the size of this effect have also been identified. These findings are very exciting as they pave the way for development of tiny transducers that could be used in the human body without posing any risk of toxicity, thus having tremendous potential for application in medicine. The work proposed in this Fellowship is centred on the development of nano-sized transducers made from phospholipids, which are the main type of fat found in membrane of biological cells. A huge area of application for the nano-transducers proposed is in medical imaging which presents a number of challenges. In practice, the nano-transducers could be used to remotely probe tissue properties and used in an imaging system to aid the diagnosis of disease. There is also a growing need for new imaging systems capable of remotely studying cells and tissues in the body to support the development of emerging therapies that use human cells to treat currently incurable conditions, such as Parkinson's disease and spinal injury, as well as chronic conditions including diabetes and heart disease. The hope is that by introducing new healthy cells into the body they will help to restore the function of injured or diseased cells. To ensure these therapies have a positive effect it is important that the location and behaviour of introduced cells are tracked once in the body. This is a challenging problem which current technologies are struggling to address. The work proposed in this Fellowship will address the above challenges. The approach that will be taken is different from other workers particularly as it will involve the development of transducers made from organic material. A major part of the proposed work will be designing and fabricating the nano-transducers. The phospholipids the nano-transducers will be composed of will be formed into bubbles called liposomes. Due to the natural link between the electrical and mechanical properties of liposomes it will be possible to use them as tiny acoustic sources. Strategies to increase the size of the acoustic signal produced will be developed based on modification of the liposome composition, shape and size. Another part of this Fellowship will be the development of a suitable imaging system using the nano-transducers that can be used to produce diagnostic images of the body. Also by controllably decorating the liposomes with specific biological molecules the nano-transducers will be able to target certain cell types enabling them to act as beacons to locate cells in the body. The final part of the work will be centred on demonstrating the capability of the new imaging system using tissue phantoms that mimic the human body. In particular, the ability to detect tumours, electrical activity in the brain and track cells used in therapy will be investigated. Overall, the success of this work will deliver a new medical imaging modality that could be implemented readily within clinical pathways at the point of care. This would have a significant impact on healthcare and enable new therapies to become available for clinical use and thus contribute to the health and wealth of society.
Protein-protein interactions mediate most biological processes and are therefore important therapeutic targets. The biological activity of a protein usually stems from only a small localised region on its surface. At the molecular level such regions often correspond to key secondary structures known as alpha-helices or beta-sheets that reside within the protein. Creating molecules able to mimic these regions while retaining their structure are attractive options for drug design. However short regions of a protein are usually unable to adopt these structures in the absence of the rest of the protein. Rather, they populate random structures that are susceptible to degradation in addition to other shortcomings such as their inability to cross biological membranes and poor bioavailability. To circumvent these issues we will collaborate with the Fairlie, a world leader in secondary structure mimetics, to create peptides that are able to form bioactive alpha-helices and beta-sheets in isolation. This will be achieved by introducing helix- or strand-inducing tethers into our growing collection of library derived peptides. Shorter constrained peptides can be derived from larger peptides known to bind with high affinity to their target. Our efforts will focus on two key areas in which we have track record: i) creating peptides to antagonise the oncogenic transcriptional regulator, Activator Protein-1. We have previously used library screening assays to derive a range of peptides capable of antagonising function. We have already worked with Fairlie to demonstrate feasibility for this approach by targeting one AP-1 partner known as cFos and shedding over 40% of the peptide in the process. Using this approach we were able to derive stable helix-constrained peptides specific for their target protein that also resisted degradation (Rao et al, PLOS One 2013). We believe that much high affinity interactions can be achieved by targeting another AP-1 component, known as cJun, where many more hydrophobic interactions required for high binding affinity can be formed. Previous related work has demonstrated that this approach can yield tethered peptides as short as five amino acids (Harrison et al, PNAS, 2010) that are able to meet many of the requirements necessary for a drug, such as high stability and resistance to biological breakdown. ii) Creating peptides capable of modulating amyloid formation. We have used library screening to derive small beta-strand peptides that bind to the Alzheimer's beta-amyloid peptide (Acerra et al, Protein Eng Des Sel 2013). We now seek to collaborate with Fairlie in creating mimetics of these short peptides that result in improved compounds that are able to circumvent many of the above issues. To achieve these goals Mason will travel to the Institute for Molecule Bioscience (IMB) at the University of Queenland on three visits over three years to further develop our collaboration with the Fairlie group. Fairlie is internationally known as a research and opinion leader in chemistry, biochemistry, pharmacology, and drug discovery. The award will permit Mason to gain new skills and techniques that can be brought back to Essex and further developed in the UK, in addition to the exchange of ideas and the further development of the collaboration. Having developed methods for stabilising alpha-helices and beta-strands in general there will be considerable scope to apply these techniques, and consequent rules for peptide and peptide mimetic design, to other peptide systems. Finally while at Queensland there will also be ample opportunity to hold seminars and meet and discuss research plans with other members of the IMB (e.g. Professors Glenn King and David Craik) who have similar interests in developing peptide-based drugs.