The control of bacterial infections is perhaps the most important achievement of modern medicine. However, the rapid emergence and spread of antibiotic resistance is fast becoming one of the major scientific and health issues of modern times. According to the "National Risk Register of Civil Emergencies", more than 80,000 deaths are estimated in the UK if there is a widespread outbreak of a resistant microbe. The development of new antibiotics is slow and difficult work but bacterial resistance is decreasing our arsenal of existing drugs. A post-antibiotic era - in which common infections and minor injuries can kill - far from being an apocalyptic fantasy, is a very real possibility for the 21st Century. The O'Neill review on antibiotic resistance sets out the global threat by highlighting that "drug-resistant infections already kill hundreds of thousands a year globally, and by 2050 that figure could be more than 10 million". Of particular concern is the mounting prevalence of infections caused by multidrug resistant (MDR) Gram-negative bacteria, in particular Klebsiella pneumoniae. This pathogen has been singled out as an "urgent threat to human health" by the UK Government, the U.S. Centers for Disease Control and Prevention, and the World Health Organization due to extremely drug resistant strains. In 2014, the percentage of MDR K. pneumoniae isolates was above 10% in more than half of all reporting European countries, including the UK. K. pneumoniae infections are particularly a problem among neonates, elderly and immunocompromised individuals within the healthcare setting, but this organism is also responsible for a significant number of community-acquired infections including pneumonia and sepsis. Very few therapeutic options are left for patients infected with MDR K. pneumoniae with additional resistance to carbapenems, and are often limited to combination therapy and to colistin. Alarmingly, recent studies have recognised that several K. pneumoniae virulent and MDR clones have access to a mobile pool of virulence and antimicrobial resistance genes; hence making possible the emergence of a MDR, hypervirulent K. pneumoniae clone capable of causing untreatable infections in healthy individuals. K. pneumoniae is exemplary of the mismatch between unmet medical needs and the current antimicrobial development pipeline. Therefore, it is an urgent priority to develop effective therapeutics based on new targets and concepts. Unfortunately, at present, we cannot identify candidate compounds in late-stage development for treatment of MDR Klebsiella infections. Rising to this health challenge, and by capitalizing on a decade of studies on K. pneumoniae infection biology of the Bengoechea laboratory, in this project we will provide solid pre-clinical evidence demonstrating that inhibition of a host protein (Src kinase) targeted by Klebsiella to ablate our defences will influence decisively the outcome of host-Klebsiella interaction thereby limiting pathogen survival. By teaming up with AstraZeneca, we will demonstrate that treatment with a company proprietary Src kinase inhibitor will favour pathogen clearance. The proven excellent safety, and tolerability of the Src inhibitor may anticipate a fast-track transition from the pre-clinical stage to further clinical clinical development hence bypassing several initial hurdles of the drug development process. Altogether, we envision that our results will encourage other academics as well as other pharmaceutical companies to follow this avenue of research to tackle the problem of lack of therapies for microbes resistant to antibiotics.

Chronic pain is a global health problem which affects approximately 30% of all adults. Current therapies have limitations in their effectiveness or side effects therefore there is an urgent need to develop more precise and effective forms of medication for individual pain causing conditions. The search for new drugs has been hampered by the lack of methods to translate early identification of promising molecules into clinically effective treatments - for example, to date much of the research needed to show drug effectiveness has been carried out in animal models which don't always show the same pain response as humans. We will address this problem by establishing and validating a laboratory model of pain causing mechanisms that was developed by Pfizer before its withdrawal from the pain treatment market and the UK as a whole. This model was developed by one of the other co-applicants on this proposal (James Bilsland) and uses nerve cells made from induced pluripotent stem cells that were themselves generated from patients who suffer from a rare but debilitating pain condition called erythromelalgia. The nerve cells that detect pain in these patients are hypersensitive - that is they produce signals that the brain interprets as pain much more easily than those of normal people. We call this phenomenon "hyperexcitability" and our aim is to identify chemical compounds which can reduce the rate ease with which erythromelalgia derived nerve cells can produce pain signals. Naturally this would be extremely valuable for erythromelalgia patients but identifying compounds to stop hyperexcitability could be much more important since there is a lot of evidence that this phenomenon contributes significantly to a lot of other pain causing conditions. In short, if we find pain killing drugs using our proposed method, they are likely to be effective against pain of many types.

Polymorphism is the ability of a compound to crystallise in more than one crystal structure. Since most drug compounds are crystalline, discovery and control of polymorphism is a key aspect of drug development and manufacturing. It is well known in this context that metastable polymorphs are usually discovered first and that stable polymorphs may make an appearance eventually (with time and money). Often, this appearance is linked to an increase in the purity of the active pharmaceutical ingredient (API). Whilst this is general knowledge shared by crystallisation scientists, we currently have a very limited understanding of the fundamental reasons behind it. In the current project we seek to utilise the above observation to our advantage. We have some experimental and computational evidence that impurities are in fact changing the thermodynamic stability of solid forms through insertion in their crystal lattices (formation of solid solutions). We seek to first test and confirm our hypothesis to then exploit this concept in order to access elusive polymorphs experimentally, be able to produce them reliably and exploit their structure and properties. Developing a deeper understanding of the impact of impurities in the formation of solid solutions and thus in the realisation of solid forms will have a significant impact in the development of pharmaceuticals, a multi-billion pounds business of great importance to the UK economy.

The COVID-19 pandemic saw the rapid development and deployment of a range of vaccine platforms. While essential to protect against severe disease, these vaccine platforms need further optimisation to provide long-term and local protection against infection including future variants. This vaccine optimisation requires an improved understanding of how a protective immune response is induced, how it is maintained, and the role of immunity in the nose and the lungs. Building on the experience our consortium amassed during the COVID-19 pandemic, we will answer some of the key outstanding questions in the field: 1.MEMORY We will delineate the mechanisms which influence the duration of protective immune responses. Improving understanding of immune memory is critical for the development and deployment of future vaccines with long-lasting protection against both pandemic and endemic pathogens. 2. LOCATION We will determine the role of the immune response in the airways, as the entry route for virus, in protection against infection. The aim is to understand if nasally administered vaccines can stop infection and onward transmission, as well as protect against severe disease. 3. PROTECTION We will define which aspects of the immune response protect against disease and how to maximise these responses. This will enable vaccine developers to focus on new vaccines that deliver improved protection. 4. DATA There exist large datasets from clinical trials and real-world studies that, if combined with the data from this programme, would generate a unique resource for understanding how vaccines work. To achieve this, we will develop an integrated data structure and open-source computational tools to integrate disparate data and maximise data usefulness. 5.IMPACT We will bolster pandemic preparedness by the training and empowerment of future leaders in vaccine development and engaging public understanding of the need for vaccines. Targeting these questions will lead to increased capability for rational, immunologically-driven vaccine development and uptake.

Over 8.5% of the world's adult population suffer diabetes. If poorly treated, diabetes leads to very high blood sugar levels which worsen the disease and lead to complications such as kidney failure and blindness, shortening life expectancy by 10 years in the case of type 2 diabetes (T2D). Pancreatic beta cells are in charge of secreting insulin in response to rises in blood sugar. Failure of beta cells to secrete enough insulin contributes to the development of diabetes. Importantly, the prevalence of high-blood sugar accelerates beta cell failure and contributes to beta cell loss by mechanisms which are not yet clear. A better understanding of the process leading to beta cell failure is vital for the development of drugs capable of stopping the development of T2D. MiRNAs are small RNA molecules that do not produce proteins themselves but are capable to reduce the rate at which other proteins (their "targets") are produced. MicroRNAs exist in beta cells that regulate important functions such as their capacity to produce and secrete insulin. Also, changes in the levels of certain miRNAs in beta cells are associated with the development of T2D. We have recently made three important findings. Firstly, when mouse and human beta cells are exposed to high levels of glucose, their levels of the miRNA miR-125b (miR-125b-5p) go up. Secondly, the introduction of additional miR-125b in the beta cells of mice causes them to produce and secrete less insulin and develop diabetes. We have also observed that reducing the amount of miR-125b in human beta cells in culture improves their capacity to secrete insulin in response to glucose. Accordingly, we hypothesize that beta cell selective inhibition of miR-125b has the potential to protect beta cell function from hyperglycaemia. Thirdly, we have seen that high levels of miR-125b lead to the appearance of enlarged lysosomes while low levels of miR-125b lead to changes in mitochondria morphology and in the content of genes related to mitochondrial function. Lysosomes and mitochondria are subcellular organelles very important for the recycling of cellular components and waste and for energy production, respectively. Thus, we hypothesize that miR-125b regulates beta cell function by modulating lysosomal and/or mitochondrial function. Both processes are essential for adequate beta cell function and are altered in diabetes. Additionally, we have demonstrated that miR-125b targets the cation-dependent lysosomal mannose-6-phosphate receptor (M6PR) which transports lysosomal enzymes to lysosomes for their adequate functioning. Nevertheless, the role of M6PR for lysosomal and secretory function in beta cells hasn't been studied. Thus, the specific aims of this proposal are to determine: 1. Whether and how selective elimination/reduction of miR-125b in beta cells prevents T2D progression 2. The role of miR-125b in lysosomal and mitochondrial function 3. The function of M6PR in beta cells To achieve these aims we will use a combination of - Mice deleted for/overexpressing miR-125b selectively in beta cells. The use of mice is necessary since maintenance of glucose homeostasis requires interplay between all metabolic tissues and therefore these experiments need to be done in the context of the whole body. - Donated human islets, modified to contain more or less miR-125b. The use of human samples is essential to ensure the translatability of our findings to the clinic. - Mouse and human beta cell lines, modified to contain more or less miR-125b or M6PR, which allow to study biological processes in detail and reduces an unnecessary use of animals. MiRNAs are novel candidates for drug targeting and our study will provide preclinical data on the potential of beta cell miR-125b inhibition for the treatment of T2D. It will also provide new fundamental insights into how beta cells work in health and disease, which, in the long term, could reveal new ways to treat diabetes.