The differing biological fates of DNA minor groove-binding (MGB) antibiotics in Gram-negative and Gram-Positive bacteria.

Antibiotics have been at the forefront of our fight against infectious disease since the 1940's. Since that time our reliance on antibiotics has been exposed by the rise of antibiotic resistant bacteria such as methicillin resistant Staphylococcus aureus (MRSA). Unfortunately, MRSA is not alone in its ability to resist the effects of antibiotics; other organisms such as Pseudomonas aeruginosa also have this ability. The World Health Organization considers solving the antibiotic resistance problem to be of global importance. One way of solving this problem is through the academic innovation of new antibiotic drugs to fight infectious disease.

We have been studying a group of compounds called MGBs that have very high activity against MRSA. Very little is known about the biological basis for this activity and we will determine the mode of action of these new drugs. We hypothesise that MGBs interfere with the ability of MRSA to control the use of its genes during infection. We will identify which genes are most potently inhibited by our new antibiotics providing us with a detailed set of targets. This information will be used in two ways. Firstly, knowledge of the targets of our drugs will help us to design new compounds that favour particular genes. Secondly, knowledge of the mode of action of a drug is important for gaining approval to use the drug in clinical trials and ultimately, the clinic.

Our previous research suggests that MGBs exhibit much better activity against organisms such as MRSA compared to Pseudomonas and E. coli. We hypothesise that this is because the latter two organisms are capable of expelling the MGBs from their cells using a system of pumps in the membrane. We will use cutting edge DNA sequencing technology to identify the resistance mechanisms of these bacteria and use this information to design new and better antibiotic MGBs to treat these infections in the future.

DNA minor groove binders (MGBs) represent a valuable chemical resource for innovation of novel antibiotics. We have previously demonstrated that MGBs are particularly active against Gram-positive bacteria such as MRSA, yet little is known about their biological mode of action. Preliminary work suggests that these compounds are not targeting DNA replication and we therefore hypothesise that MGBs have a global effect on transcription. We will use RNA-seq to monitor the abundance of all Staphylococcus aureus transcripts in the presence of our most active MGB antibiotic. We will take particular care to identify any links between the affected transcripts in terms of the GC content at those positions in order to identify and sequence specificity of the compound. Close attention will also be paid to any global stress responses that are induced by MGB treatment because this will reveal further details of the mode of action.

There is a major disparity between the activities of MGB antibiotics in Gram-positive and Gram-negative bacteria. Our preliminar data suggests that efflux pumps such as the Mex systems of Pseudomonas aeruginosa play a major role in Gram-negative resistance. P. aeruginosa strain is predicted to encode 447 membrane transport proteins and it would be incredibly laborious to screen these mutants systematically. For this reason we will use a tn-seq based approach to screen an entire non-redundant mutant library of PA14 in the presence of MGB compounds. This methods has the great advantage that it will rapidly identify a whole range of chaperones and regulatory systems as well as efflux pumps associated with Gram-negative resistance. We will use this data to prepare a smaller 'sub-library' of mutants that can be cheaply and rapidly screened in the future and to design new compounds that inhibit their own efflux. Newly synthesised compounds will be produced and tested for anti-Gram-negative activity.

The research program presented here will provide the mode of action of a new antibiotic that is effective against major nosocomial pathogens such as Clostridium difficile and Staphylococcus aureus. This drug is at an advanced stage of clinical and industrial development (see letter of support from MGB BioPharma). As such, this work will benefit society through the treatment of infectious disease. According to the Public Health Laboratory Service, the total cost to the NHS of treating nosocomial infections is estimated to be £1 billion per year. These infections lead to longer hospital stays associated with otherwise routine clinical procedures and are estimated to cost the UK economy between 3 and 11 billion per annum. A similar pattern is observed in other developed countries in North America and Europe. Therefore, improvements to the arsenal of drugs to treat these infections will benefit the individual patient as well as the NHS and the UK economy.

The main industrial beneficiary of the work proposed below is MGB BioPharma (see letter of support). MGB BioPharma is the industrial license holder of the anti MRSA drug MGB-BP3 and is currently driving the progress of this drug through pre-clinical trials. As MGB-Biopharma move closer to commercialising this drug, a better understanding of its mode of action will enhance their case and ease the regulatory burden. Because MGB BioPharma is currently at an advanced stage of the regulatory approval process for MGB-BP3 we expect real benefits to be produced within 3-5 years.

This work also aims to develop an understanding of species specificity of MGB antibiotics by studying the fundamental biological basis of MGB resistance in Gram-negative bacteria. This work will lead directly to novel MGB antibiotics targeted against organisms such as Pseudomonas aeruginosa and provide MGB BioPharma with a new pool of commersialisable compounds. Our work will enhance and secure the future of this UK based SME and we anticipate these benefits to be realised in 5 - 10 years time.

Strathclyde is committed to the enhancement of skills in the knowledge-based economy and this project is no exception. We firmly believe that the modern molecular microbiologist should be able to use next-generation sequencing instruments AND analyse the data produced. This type of work has previously been split between several specialists but we believe in equipping the PDRA on this project with the skillset that spans the entire process. The PDRA will also be encouraged to take part in schemes such as Biotechnology YES and internally provided training courses to enhance their understanding of business and entrepreneurship.