Predicting enzyme metalation to identify new therapeutic targets in infectious diseases

Microbial metalloenzymes defend against host stress responses and synthesize nutrients required for pathogenesis. Metalloenzymes unique to microbial metabolism are therefore attractive targets for therapeutic intervention, but inhibitor discovery requires knowledge of the physiological metal cofactor. Critically, the biologically important metal does not always bind the enzyme of interest most tightly when studied in the test tube, a consequence of the Irving-Williams series of transition metal-ligand stabilities. This apparent paradox is overcome in cells by buffering metal availability to differing levels. We have recently developed the tools to determine these buffered levels and thereby overcome the challenge of identifying the physiological metal cofactor of a variety of metalloproteins.
Clostridium difficile, the leading cause of hospital-acquired diarrhoea, undergoes sporulation as an essential part of its transmission cycle. Indeed, spores are the infective agent, responsible for infection as well as recurrency. Global regulation of sporulation is known but the requirement for and supply of nutrients, including metals, during this process is poorly studied. Sporulating pathogens therefore offer unique metalloenzyme therapeutic targets that, when inhibited, would limit the spread of infection due to diminished sporulation.
To determine the correct metal cofactors of Clostridium enzymes essential for sporulation, metal availabilities will first be determined using our recently developed approach. Clostridium metalloregulatory proteins will be overexpressed and purified to enable determination of metal and DNA-binding affinities using spectroscopic methods. These affinity values will be used to define physiologically available metal levels within the cell. Then, metal affinities of purified Clostridium enzymes will be determined to identify the physiological metal cofactor, ie, the one that matches calculated availability. A complementary line of investigation will examine the relationship between metal availability and nutrient supply on Clostridium sporulation efficiency by focusing on known biosynthetic pathways containing one or more key metalloenzymes.

The CDT has five primary beneficiaries:
The CDT cohort
Our students will receive an innovative training experience making them highly employable and equipping them with the necessary knowledge and skillset in science and enterprise to become future innovators and leaders. The potential for careers in the field is substantial and students graduating from the CDT will be sought after by employers. The Life Sciences Industrial strategy states that nearly half of businesses cite a shortage of graduates as an issue in their ability to recruit talent. Collectively, the industrial partners directly involved in the co-creation of the proposal have identified recruitment needs over the next decade that already significantly exceed the output of the CDT cohort.
Life science industries
The cohort will make a vital contribution to the UK life sciences industry, filling the skills gap in this vital part of the economy and providing a talented workforce, able to instantly focus on industry relevant challenges. Through co-creation, industrial partners have shaped the training of future employees. Additional experience in management and entrepreneurship, as well as peer-to-peer activities and the beginning of a professional network provided by the cohort programme will enable graduates to become future leaders. Through direct involvement in the CDT and an ongoing programme of dissemination, stakeholders will benefit from the research and continue to contribute to its evolution. Instrument manufacturers will gain new applications for their technologies, pharmaceutical and biotech companies will gain new opportunities for drug discovery projects through new insight into disease and new methods and techniques.
Health and Society
Research outputs will ultimately benefit healthcare providers and patients in relevant areas, such as cancer, ageing and infection. Pathways to such impact are provided by involvement of industrial partners specialising in translational research and enabling networks such as the Northern Health Science Alliance, the First for Pharma group and the NHS, who will all be partners. Moreover, graduates of the CDT will provide future healthcare solutions throughout their careers in pharmaceuticals, biotechnology, contract research industries and academia.
UK economy
The cohort will contribute to growth in the life sciences industry, providing innovations that will be the vehicle for economic growth. Nationally, the Life Sciences Industrial Strategy Health Advanced Research Programme seeks to create two entirely new industries in the field over the next ten years. Regionally, medicines research is a central tenet of the Northern Powerhouse Strategy. The CDT will create new opportunities for the local life sciences sector, Inspiration for these new industries will come from researchers with an insight into both molecular and life sciences as evidenced by notable successes in the recent past. For example, the advent of Antibody Drug Conjugates and Proteolysis Targeting Chimeras arose from interdisciplinary research in this area, predominantly in the USA and have led to significant wealth and job creation. Providing a cohort of insightful, innovative and entrepreneurial scientists will help to ensure the UK remains at the forefront of future developments, in line with the aim of the Industrial Strategy of building a country confident, outward looking and fit for the future.
Institutions
Both host institutions will benefit hugely from hosting the CDT. The enhancement to the research culture provided by the presence of a diverse and international cohort of talented students will be beneficial to all researchers allied to the theme areas of the programme, who will also benefit from attending many of the scientific and networking events. The programme will further strengthen the existing scientific and cultural links between Newcastle and Durham and will provide a vehicle for new collaborative research.