Modulation of antibiotic resistance and protein synthesis by disrupting Elongation Factor G dynamics

Antibiotic resistance is a serious public health concern; the Chief Medical Officer for England warned it poses a 'catastrophic threat' on a par with climate change or terrorism and deaths related to antibiotic resistance are predicted to outnumber those from cancer by 2050. Without concerted efforts, we face a future in which routinely treatable infections may become fatal due to a lack of effective antibiotic treatments. As few new antibiotics are becoming available it is important to overcome resistance to existing antibiotics to extend the usefulness of these important treatments. To do this we must understand how the proteins involved mediate resistance. This work aims to understand the mechanism of resistance to an important clinical antibiotic, fusidic acid (FA), and identify key regions of the proteins involved that control how they act. This could provide resources for the development of drugs to overcome this resistance and rejuvenate the usefulness of this important antibiotic.

FA is used against infections by the bacteria Staphylococcus aureus and is one of few remaining oral antibiotics active against the hospital 'superbug' MRSA. FA interacts with a protein called EF-G (an important part of the machinery for making proteins) and prevents it from working, meaning bacteria cannot make proteins and so cannot grow. Resistance to FA has increased dramatically in recent years either by the interaction of another protein, FusB, with EF-G that rescues it from the effects of FA, or mutations in EF-G itself. The interaction between FusB and EF-G has recently been studied, showing FusB causes long-range changes in EF-G that produce motions in EF-G important for causing FA resistance. I have new data showing that I can disrupt these motions by altering EF-G and that when I do, the proteins become less resistant to FA. I aim to determine which parts of EF-G are important in controlling these motions to try to find key areas of EF-G that could act as targets for the development of drugs that can stop FA resistance and so extend the usefulness of this antibiotic. I will make a series of changes to EF-G and monitor the effects of those changes using structural investigations of motions in EF-G by a technique called nuclear magnetic resonance (NMR). I will then study what effects these changes have on the ability of FusB to cause FA resistance, identifying important areas of EF-G that control its response to FusB. I will use knowledge of these important areas to see if changing them in EF-G that is already resistant to FA without needing FusB can prevent it from producing FA resistance too, suggesting any areas that might control both types of resistance. To complement this, I will study whether similar motions in EF-G are important in the function of EF-G in making proteins within bacteria to try to get a better understanding of how this protein works. This could provide further information for drug discovery studies to try to stop this essential protein from working, providing a potential target for antibiotic development studies. These studies will allow us to understand how bacteria become resistant to this important antibiotic, providing information that can be used to design new antibiotic treatments to bypass this resistance. They also help us to understand the role of EF-G in the essential process of making proteins, which may show more potential drug targets for development of new antibiotics.

With the increase in antibiotic resistance and few new antibiotics being discovered, it is important to use knowledge of current resistance mechanisms to develop drugs that bypass resistance or to design drugs that can be added to antibiotics to overcome the resistance. Understanding the structural basis of antibiotic resistance can lead to the development of such drugs that can be administered with the antibiotic to overcome the existing resistance, allowing us to continue to use the antibiotic to treat infections.

Fusidic acid (FA) is an important antibiotic used to treat Staphylococcal infections, including MRSA infections. FA blocks bacterial protein synthesis by binding to elongation factor G (EF-G) and preventing its release from the ribosome. Resistance to FA is rising due to the expression of FusB-like proteins that bind to EF-G and overcome FA-mediated inhibition. FA resistance is also mediated by amino acid substitutions in EF-G, some of which are likely to perturb FA binding but which are spread widely in the protein structure and are likely to mediate resistance by other mechanisms. My recent EF-G(domains 3-5):FusB structure showed FusB allosterically induces a change in the conformational flexibility of EF-G and new preliminary data shows that this change in conformational flexibility is the means by which FusB confers FA resistance as disrupting it causes an increase in FA sensitivity in the presence of FusB.

Changes in FusB-induced dynamics in EF-G in response to mutations within EF-G will be characterised using NMR to determine which residues are important in controlling the change in dynamics and transmitting the allosteric response through the protein. The effects of mutations perturbing dynamics within EF-G on FA affinity will be elucidated to identify any key sites that could act as druggable targets for future development of FA resistance inhibitors. Differences in dynamics and EF-G structure in response to the GTP/GDP bound state will then be studied to determine if EF-G dissociates from the ribosome following translocation using a similar mechanism to that employed by FusB. This will provide further understanding of the mechanism of ribosome translocation and may suggest alternative target sites for future drug development studies.

This work will allow the detailed characterisation of FA resistance, identifying potential druggable sites for the development of inhibitors of resistance that could extend the usefulness of this important antibiotic.

Potential benefits of the proposed research outside of the academic sector are as follows:

(i) Benefits to industry. - The research contained in this proposal will provide detailed information on the molecular mechanism(s) of fusidic acid resistance and will identify key amino acids controlling changes in conformational flexibility and transmission of the allosteric response that control fusidic acid resistance. This will identify any hotspots that could become druggable targets for inhibitors of the antibiotic resistance mechanism. This information is expected to be of significant use for structure based drug design strategies to develop novel antibiotics or adjuvants to be administered with fusidic acid to overcome resistance and therefore rejuvenate the usefulness of this important antibiotic. Fusidic acid is clinically important and is now off patent and so development of inhibitors of fusidic acid resistance could be an attractive target for commercial drug development programmes. Therefore, these studies will benefit industry by providing the scientific knowledge necessary to drive such commercial drug design initiatives to potentially develop new drug treatments.

(ii) Social and economic benefits. - In the short term, this work will build UK expertise and capability in the fields of antibiotic resistance and NMR spectroscopy. The research will provide training and experience for a new member of research staff. In the longer term, this work aims to deliver an understanding of the mechanism of resistance to the important antibiotic, fusidic acid, and identify key areas that control this mechanism with the aim of promoting the use of this knowledge for development of novel adjuvants that can be co-administered with fusidic acid. Such adjuvants have been successfully used in rejuvenation of the clinical use of beta-lactam antibiotics through co-administration of beta-lactamase inhibitors. This work is therefore aimed as an initial step towards producing strategies to address the increasing problem of drug resistant infections which now represents one of the most pressing areas of unmet medical need. Indeed, the World Health Organisation has declared antibiotic resistance one of the greatest threats to human health, food security and development. Successful delivery of this aim would therefore have a significant positive impact on health and the quality of life in the UK and worldwide.