A protein called Ras has, for many years, been an extremely important therapeutic target in many types of cancers, yet the ability to target Ras with drugs is still elusive. Under normal conditions Ras acts as a switch that turns on and off, regulating major cellular processes such as growth, division and migration. In a high proportion of cancers the Ras switch is broken and remains in a 'on' state causing uncontrolled cellular growth. This process is often a result of Ras mutations causing the protein to avoid the normal regulatory mechanisms which usually turn it off or regulate it being on in the first place. As part of an Innovate feasability grant in collaboration with a company (Tecrea) our group generated reagents, termed Affimers, that blocked Ras function. Interestingly the reagents can either block Ras from being turned on in the first place or specifically target mutant Ras and turn it off. This represents a very exciting development in understanding the ability to specifically target mutant Ras proteins and could aid the design of drugs to mimic the Affimers or be used as drugs per se. However, the aim of this proposal is to determine how the Affimers are inhibiting Ras function, to try improve on this ability by modifying the Affimers and test their ability to modulate Ras function in cells.

A major challenge for the use of proteins as biopharmaceuticals lies in their inherent instability. Despite the widespread appreciation of this issue, whether instability arises from thermodynamic instability, kinetic instability and/or the propensity of proteins to aggregate has not been explored in any systematic way. Here we propose to investigate the origins of instability of antibody domains using a wide range of biophysical methods, with the overall goal of determining which of these fundamental features is key to determining instability and, thereby, to explore new routes to evolve or enhance biopharmaceutical 'shelf-life'. The proposal, therefore, clearly falls in BBSRC's remit and strategic plans, spanning many target areas as outlined below. We will focus our studies on antibody light chains, including both the constant and variable domains, and sequences known to be stable, or to be aggregation-prone. (i) Thermodynamic parameters will be obtained from equilibrium denaturation experiments (using DSC, fluorescence and/or CD as probes); (ii) kinetic parameters will be measured using stopped flow spectroscopy and amide hydrogen exchange monitored using NMR and/or mass spectrometry and (iii) instability due to self-association will be monitored using light scattering and analytical ultracentrifugation). We will also explore the effect of commonly used small molecule additives to tailor each of these properties for different antibody sequences, and will explore the use of novel reagents as stabilising agents that target the different aspects of instability. In addition, we will explore the possibility of using protein engineering to develop new antibody domain sequences that maintain function, yet are tailored specifically to have enhanced stability and solubility in vivo. Identifying such mutations is immensely challenging, however, because the origins of stability in vivo are complex, multi-factorial and inter-related (depending both on the physicochemical properties of the protein of interest, but also its interactions with the cellular milieu). To achieve this, we will use the power of directed evolution to create new protein sequences selected to possess enhanced stability in vivo, using a novel strategy recently developed in SER's laboratory (with Bardwell, Michigan) in which the in vivo stability of a protein is directly linked to antibiotic resistance (Foit at al, currently under revision at Molecular Cell). The approach requires no prior structural or functional knowledge and permits the evolution of protein stability independently of functional constraints. By combining the sequences evolved, with the additives above, our ultimate goal is to develop new, informed routes to enhance stability that should be of general utility across the biopharmaceutical industry. In addition to being of immense practical utility, the ability to evolve protein stability in vivo may indicate how different evolutionary pressures have interacted to produce today's protein sequences. The project will take advantage of the complementary skills of the academic and industrial collaborators. Prof Radford (Astbury Centre, Leeds) has an established track record in the biophysical characterization of protein stability, folding and aggregation spanning 22 years. Professor Smith (Avacta) brings experience in high throughput analysis of protein stability based on the newly developed instrumentation in Avacta and knowledge of protein additives as stabilizing agents in the biopharmaceuticals industry. The collaboration will thus open new doors for research opportunity that will generate new knowledge of immense value both from fundamental and applied viewpoints.

The applicants have developed a novel technology based on low-cost semiconductor and molecular technologies that is able to detect minute protein traces, enabling rapid in-vitro detection of multiple biomarkers, including disease biomarkers, at low cost and without the need for complicated assays. This disruptive technology has the potential to impact upon a number of large, growing markets. Our technology offers easy-to-use, multiplexed electronic detection of biomarkers and has the potential to be used in hundreds, if not thousands, of diagnostic applications in multiple sectors including healthcare, veterinary, drug development, security and agrotech. More information is available on our website www.etect.co.uk.

A famous biochemist, Arthur Kornberg, in his book "For the Love of Enzymes" once said 'DNA and RNA are the script, but proteins are the actors'. Proteomics, the study of the actors, (whether in soliloquy or in crowd scenes) has terrific potential in diagnostics, in the analysis of new disease biomarkers, in understanding the fundamental ways by which the 'intent' of the genes is realised. At present, the global study of proteins (proteomics) is lagging behind our understanding of genomes and RNA, and without radical new technical approaches, taking the best of the analytical capability and coupling it to new methods of sample delivery, the gap is likely to widen. The challenges are several. First, a true global proteome analysis has to be able to deal with a highly complex mixture of proteins, some present in huge quantities, others at vanishingly low levels. This will require a degree of normalization, in which low abundance proteins are brought to the analytical step in sufficient amounts for analysis, and high abundant proteins are non-selectively sampled. Secondly, current proteomics is still predominantly based on prior digestion of proteins to multiple smaller peptides using an enzyme (trypsin) derived from the gut. This not only increases analyte complexity about 50 times but also conceals much of the subtlety of the protein world (just as a pile of bricks cannot inform about the structure of the the many building types and variants that could have been made from those bricks). A future solution should be based on protein-level analysis - architecture is less about the study of bricks than it is the exploration and celebration of the entire structures that the bricks are assembled to create. Finally, we deliver peptides slowly (1-4h per sample) by rather troublesome chromatography. Alternative approaches to protein-level delivery are required. I propose that we should plan to analyse a proteome without the complication of digestion or of chromatography. This poses new challenges, because we cannot expect the mass spectrometer to be able to analyse a whole proteome at once (it is just too complex). I therefore wish to devise an entirely new approach to proteome analysis based on delivery of a small number of proteins at any one time to the analytical platforms. The industrial collaborator has invented new types of genetically altered proteins ('Affimers', because they have a high affinity for selected target proteins) that are capable of selectively binding and fishing out a few proteins at a time. With appropriate analytical instrumentation, we should then be able to deliver the payload (proteins) in such a way that we can analyse them directly, capturing all of the complexity of the protein world - the architect's view. In the longer term, engineering solutions to payload delivery could make this the preferred approach to proteome analysis.

The use of drugs as medicines has made an enormous contribution to human health with the drug discovery process being directed to most aspects of human disease. However, all drugs are associated with variability in response: that is some patients do not respond to drugs, while others develop side effects or adverse drug reactions (ADRs). This may be related to patient factors (genetic or environmental) or differences in their disease. The overall burden caused by differences in the way patients respond to drugs is large to both the NHS and Industry. For example, with ADRs, we have shown that at least 6.5% of all adult admissions to hospitals are due to ADRs and that approximately 15% of inpatients suffer an ADR during hospitalisation. Extrapolated nationally, ADRs are thought to cost the NHS in England in excess of £637 million annually, or approximately £5000 per hospital bed per year. This is a conservative estimate and is likely to be much higher. Our findings in the UK have been replicated in many countries showing that ADRs are a global health issue. Our application focuses on the MRC Centre for Drug Safety Science (CDSS) which has an international reputation. The patient is at the centre of the work we undertake. We have developed an infrastructure that allows pre-clinical and clinical scientists to work side-by-side using cutting-edge technologies to analyse well-defined clinical samples. In this application, we request state-of-the-art technologies to build upon this infrastructure and produce a step-change in the area of stratified medicine - that is, to identify the best treatments for patients based on profiling their disease, genetic and environmental factors. Our aim is to develop a comprehensive resource, based on our existing expertise and experience and the technology platforms, to allow for assessment of how individuals vary in drug responses, how diseases differ between individuals, and how this relates to variation in clinical outcomes. We will do this through investigation of different "experimental" systems ranging from single cells in test-tubes to experimental studies in man, to careful clinical observation of patients in clinical settings. This will facilitate translation of findings in the laboratory to clinical care (bench to bedside), but importantly lessons learnt in clinical settings will also be investigated further in the laboratories (bedside to bench), so that we can learn more about disease processes.