Development of antimicrobial peptides against Gram-negative antibiotic resistant pathogens

The underuse and overuse of antibiotics has resulted in Gram-negative bacteria becoming resistant to known antibiotics. Although this is a global problem, the lack of new antibiotics and alternative therapy is of concern especially in SA, where patients with a weakened immune system, such as those with HIV, are highly susceptible to infection. The diagnosis of Gram-negative infections is often difficult, especially when these occur deep in the body. The resistance to antibiotics used to treat these infections makes the situation worse as the infection progresses undetected and so both new antibiotics and new ways of monitoring the success of treatment are urgently needed.

This is a global crisis and only through an international collaborative scientific effort, will new antibiotics be discovered. An important aspect of this process is to build capacity in peptide based antibiotic drug discovery. A focus of such an endeavour is to provide scientists from historically disadvantaged backgrounds and institutions in SA the opportunity to be leaders in this field of research. Through this process, the training of the next generation of scientists is essential to ensure a continued success to find novel approaches to combat the perpetual threat of evolving bacterial infections, especially in the context of unique health care challenges in South Africa.

One such approach is based on using antimicrobial peptides (AMPs), nature's own antibiotics which have remained effective throughout evolutionary history, but which have yet to make a breakthrough into clinical use. AMPs are key components of the innate immune system of a huge variety of living organisms, including humans, and have the ability to eradicate infection. Now, to show that AMPs can be developed as antibiotics for therapeutic application in patients we will learn how to modify them to increase their safety and stability. We will also ensure that they are more likely to work in patient by testing them in conditions that match more closely the patient infection setting. Existing methods of laboratory antibiotic testing are deficient as they often under- or over-estimate the potency of different types of antibiotic, most notably AMPs .

As well as treating patients, we want to use AMPs to monitor their recovery or tell us when other antibiotics should be given. We will adapt imaging techniques, recently developed to study brain activity or find tumours and reveal the success of radio- and/or chemo-therapy. AMPs can be modified to carry a radioactive tracer and will circulate around the body before homing in on the bacteria causing infection. The tracer that is attached is used for either positron-emission tomography (PET) or single-photon emission computed tomography (SPECT) where a relatively low, and hence safe, dose of gamma rays is emitted. The intensity and location of the PET or SPECT signal can be mapped in the body in three dimensions to give the location and extent of any infection and to signal when, e.g. a localised infection starts to spread around the body. Since AMPs selectively target bacteria radiolabeling can provide a rapid, noninvasive method for infection imaging and with the correct antibiotic treatment, rapid patient recovery with reduced risk of antibiotic resistance development.

Through Newton funding a UK and SA collaboration will be established to accelerate the innovative discovery of new AMP-based antibiotics. Through increased mobility and exchange of UK and SA researchers from UP, NWU and SMU, links between researchers in both countries will be strengthened and new partnerships will be developed with the possible establishment of a global pre-competitive drug discovery consortium. This process has a further benefit and that is the strengthening of the strategic relationship between the UK and South Africa.

The main aim of this study is to accelerate antibiotic drug discovery with specific focus on AMPs, as novel antibiotics and as infective selective tracers for non-invasive molecular (PET or SPECT) imaging. Rational design of peptide analogues will follow a mechanistic understanding of unique AMPs sourced from African species, which uses a combination of systems and both steady-state and time-resolved biophysical methods (MD simulations, CD, solid-state NMR, patch-clamp, fluorescence spectroscopy, electron microscopy). Focusing on Klebsiella pneumoniae and Pseudomonas aeruginosa - Gram-negative bacteria which grow as biofilms and are the most common pathogens that cause lower lung infection and keratitis in neonates, children and immune compromised individuals - we will develop novel combinations of existing and newly adapted approaches to evaluate antibacterial potency. This is based on recent research at King's and by others that shows that membrane active antibiotics, notably AMPs, are particularly sensitive to bacterial culture conditions. We will therefore adapt both in vitro (bespoke broth microdilution, shear flow, hollow fibre) and ex vivo (porcine cornea or lung) antibiotic susceptibility testing to make these more representative of each infection setting. We will apply NMR metabolomics and RNAseq to define bacterial metabolism and responses to AMPs in each system to better understand features of AMPs that will lead to therapeutic success. Therefore, both potency and toxicity will be evaluated in conditions that more accurately represent the circulatory system as well as, lung and corneal mucosa. The ability of these peptides to specifically target Gram-negative bacteria also supports their development as a component of a radiolabeled tracer. Using e.g. tris(hydroxypyridinone) ligands for 68Ga or 99mTc chelation, chelator-AMP conjugates will be synthesised and used to localize infections and to monitor response to antibiotic treatment.

Drug resistant Gram-negative infections have become a global problem with very few new drugs being developed for treatment. Through innovative collaborative research between the UK and SA new peptide-based antibiotics and diagnostic strategies to identify these infections are being developed. The first impact of this research is the development of a drug discovery pipeline that includes SA and UK research teams to increase the rate of throughput of candidate peptides, from the laboratory bench to the pre-clinical phase of testing. Gram-negative bacteria targeting peptides will be developed as antibiotics and following radiolabeling will be used to detect and monitor infection with PET.

Besides an increase in research capacity, the investment in the next generation of young scientists will leave a long-lasting legacy and will have the greatest impact. These scientists will through the application of their knowledge and skills, be in the forefront of drug discovery and through scientific endeavor will develop and find innovative ways to prevent and treat drug resistant infections.

The long-term beneficiaries in SA are neonates, children and immunocompromised individuals where a new generation of antibiotics will reduce the risk of serious infections, neonatal sepsis and health-care associated infections. In these patients, it is often difficult to identify the site of infection, non-invasive diagnostic methods have the potential to localise sites of infection and a further advantage early treatment reduces the risk of difficult to treat biofilms from developing. This is most probably where the biggest societal impact will be made and the subsequent economic benefits that will flow out of this are reduced hospital stays and pressure on limited healthcare resources that is being experienced in SA.

The project forms part of a wider programme of research that will have wide-ranging economic, animal and public health benefits in the longer term. These will arise from the creation of new and enduring classes of antibiotic, a better understanding of how and why successful bacterial therapy proceeds, the synergistic interaction of exogenous antimicrobials with the host innate-immune system and new techniques and tools to monitor hospital acquired infections and use antibiotics more appropriately. Notably, the O'Neill reports state that "...failing to tackle drug-resistant infections will cause 10 million deaths a year and cost up to US$ 100 trillion by 2050." The outcomes of this project will make a substantial contribution to mitigating this threat by accelerating antibiotic drug, diagnostic and/or theranostic discovery. This will stimulate the discovery of new antibiotics and antibiotic combinations which will be effective in the face of bacteria carrying existing resistance determinants and help in developing new ways of monitoring and reacting to the emergence of resistance to antibiotics and/or antiseptics.