Extending bicyclomycin treatment of multi-drug resistant Gram-negative pathogens

Antibiotics have saved millions of lives from infectious diseases and are arguably the greatest medical discovery of the 20th century. Unfortunately, one of the biggest threats to public health in the 21st century is the rise of multi-drug resistant bacterial infections. This rise has been caused by the improper use of antibiotics in medicine and agriculture combined with a shortage in the discovery of new types of antibiotics. This has prompted the World Health Organisation to warn that "with a dearth of new antibiotics coming to market, the need for action to avert a developing global crisis in health care is increasingly urgent", and the UK's Chief Medical Officer, Prof. Dame Sally Davies, to declare that "we are also not developing new drugs fast enough". This is a global problem that requires global action, particularly for the treatment of multi-drug resistant Gram-negative bacterial infections. Gram-negative bacteria are naturally resistant to many antibiotics used clinically, and have evolved to be resistant towards most other antibiotics following years of treatment with those medicines.

There are various approaches to tackle this growing problem, including re-purposing "old" antibiotics. These are drugs that were thoroughly tested for efficacy and safety in human clinical trials, but were not then widely used for a variety of reasons. For example, better broad-spectrum alternatives may have been available at the time. The rise of multi-drug resistance means that these molecules may now be very useful, especially because their lack of clinical use means that there has not been an opportunity for resistance to develop in disease-causing bacteria. Additionally, new experiments can sometimes reveal antibacterial activities that were not identified in earlier research. This is the case for bicyclomycin, an old antibiotic that had previously been shown to have moderate bacteriostatic activity towards Gram-negative bacteria. "Bacteriostatic" means that the antibiotic stops bacterial growth, but does not actively kill the bacteria, which can lead to the persistence of an infection. Excitingly, recent work has shown that bicyclomycin can actually kill bacteria ("bactericidal") when it is used alongside another bacteriostatic antibiotic. This unexpected activity makes bicyclomycin a highly promising antibiotic for the treatment of Gram-negative bacterial infections when used in combination with another drug.

Therefore, we propose to carry out further work to determine whether bicyclomycin can be widely used in the clinic. This will include testing more accurate models of infection for this novel activity and identifying new compounds that can be used alongside bicyclomycin to stop resistance developing. Bicyclomycin is a molecule that is made naturally by non-pathogenic soil bacteria. Natural products such as this are produced by the action of a series of enzymes (proteins), which are encoded by genes (DNA) in the bacterial genome. Thus, we aim to discover the genes that are responsible for bicyclomycin production. This discovery will allow us to make modifications to the pathway to make more of the compound, which will enable its study in infection models. We can also modify the pathway to produce new versions of bicyclomycin, which might have better activity than the original compound or overcome resistance mechanisms.

Our research programme aims to repurpose an old antibiotic, bicyclomycin, for treatment of multi-drug resistant (MDR) Gram-negative infections. This stems from recent work demonstrating its ability to rapidly kill Gram-negative bacteria when used in combination. Bicyclomycin is a Streptomyces natural product that was discovered in the 1970s and has a narrow bacterial spectrum, poor oral absorption, and little lethal activity. Consequently, this compound, along with many others, was set aside in favour of more active agents that were available at the time.

Bicyclomycin has now become a promising candidate for redevelopment as members of the research team have shown that combining bicyclomycin with a bacteriostatic inhibitor of transcription (e.g. rifampicin) or translation (e.g chloramphenicol) converts bicyclomycin from a bacteriostatic agent to a highly lethal agent against multiple Gram-negative species. This discovery of bicyclomycin lethal synergy was surprising, because with most, if not all, other antimicrobial combinations, bacteriostatic inhibitors of gene-expression antagonise rather than stimulate the lethal activity of bactericidal agents. We have evidence that indicates that in the absence of a suitable partner antibiotic, a protein that protects against the lethal activity of bicyclomycin can be expressed. A small-molecule inhibitor of this protective protein combined with bicyclomycin would create a novel, highly lethal combination therapy for MDR Gram-negative infections. To achieve this we need to: (1) determine whether lethal synergy is observed in an in vivo model of infection; (2) establish a robust, high-titre platform the production of bicyclomycin by its natural Streptomyces producer, and develop methods to produce novel derivatives by both pathway engineering and synthetic chemistry; and (3) characterise the protective mechanism using proteomics and subsequent gene deletions to inform a screen for inhibitors of this process.

The main beneficiaries of the proposed research will be the academic research community (see academic beneficiaries) who will benefit from the result of proposed work across multiple scientific disciplines, including infection studies, biosynthesis, medicinal chemistry and structural biology. This will result in the generation of various models we build from this work (such as a model of lethal synergy, and a model of bicyclomycin biosynthesis). This will be published in top class open access journals and will be presented at national and international conferences across disciplines. The project will establish a new collaboration between groups at the John Innes Centre (JIC) and Xiamen University, and the researchers involved in this project (UK PDRA, Chinese PhD students and research assistants) will benefit from exposure to a multidisciplinary project that encompasses a variety of research areas that are valuable for 21st century antimicrobial drug discovery.

A public consultation by JIC (coordinated by Ipsos MORI) determined that antibiotic resistance is seen by the general public as one of the major challenges facing both the UK and the developing world. It is therefore vital that we effectively communicate our research to the public so they can understand what we are doing to develop new approaches to overcome AMR. We will employ various outreach strategies that have previously proven to be highly successful. This includes our 'Antibiotic Hunters' exhibit about antibiotic discovery that was first prepared for the Great British Biosciences Festival in 2014. The bicyclomycin story will make a great addition to this exhibit, which allows the public to directly speak to members of the research team in a relaxed environment. This will be presented at local and national public outreach events and at local schools and hospitals. We will also reach the public by engaging with the media via our press office when we publish the results of this study. This will ensure there is impact from this research beyond academia.

The major mode of outreach for this project involves the SAW Trust (www.sawtrust.org, reg. charity no. 1113386), which is a science education initiative based on the Norwich Research Park that uses art and creative writing to effectively communicate a diverse range of cutting edge research topics in schools. It has already been a great success in Shanghai, where it was introduced into schools by the Xuhui Education Bureau. Since the first Xuhui SAW workshop in 2012, the programme now involves 18 schools and kindergartens, 140 teachers and around 50 social volunteers. The SAW learning style has proven to be very popular with Chinese teachers and creates opportunities for parents to work alongside teachers to deliver valuable curriculum enrichment activities in schools. This prior Chinese experience means that SAW is ideally placed to implement an antibiotic-themed project, which will be initially implemented at a middle school affiliated with Xiamen University, and would then be shared widely to other schools as an important learning resource. The project will be designed with scientific input from the research team and will be tested in a UK school before being translated and transferred to China. Both the UK PI and co-PI have been involved in multiple SAW activities in the UK relating to antibiotics.

There is also scope for the discovery and development of novel IP in the form of new versions of bicyclomycin, improved production methods, and co-administration strategies. This has the potential for economic benefits in additional to the obvious social benefits that come with developing new treatment modalities for antibiotic resistant bacterial pathogens. As appropriate we will continue to liaise closely with our technology transfer offices to protect and exploit any novel IP that arises from this work.