Understanding Peptide Antibiotics at the Single Molecule Level

Antimicrobial peptides are agents of the most ancient defence systems; they assist multicellular organisms in fighting against microbes. Most membrane active peptides act by affecting lipid bilayer integrity either by disruption or pore formation. Our knowledge of the pores formed by antimicrobial peptides is limited as their small size and heterogeneity makes them difficult to study using many conventional techniques. Recent advances in methods to detect individual molecules give us the opportunity to explore the mechanism of action of these peptides in greater detail. I propose to study the mechanism of action of several membrane active peptides found in nature which bind to and insert into lipid bilayers to form pores. I will study three groups of pore forming compounds by applying simultaneous single molecule fluorescence and single channel electrical recording measurements to image the pores they form.1. Alamethicin and magainin II; the two archetypal pore forming peptides.2. Equinatoxin II; an actinoporin, part of the host defence mechanism of the sea anemone Actinia equine.3. Daptomycin; a lipopeptide antibiotic in clinical use.At a time of increasing bacterial resistance these peptides are of great interest as potential antimicrobial drugs. Knowledge of their mechanism of defence against infection will also aid in the development of novel, synthetic antimicrobial agents. These compounds may augment immunity, restore potency or amplify the mechanisms of conventional antibiotics and minimise antimicrobial resistance mechanisms among pathogens.

The beneficiaries of this research include pharmaceutical companies, the NHS and ultimately the general public. How will they benefit from this research? In order to respond quickly and adequately to the invasion of pathogens, organisms have developed antimicrobial peptides (AMPs) as a first line of defence. In recent times we have seen the emergence of multi-drug resistant bacteria, largely due to the mistreatment of infection and over-prescription of antibiotic drugs. AMPs may present valuable alternatives to existing antibiotics. Knowledge of the mechanism of action of AMPs on a molecular level will feed into the development of novel antimicrobial drugs that are active against the so called super-bugs such as, for example, Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA). A deeper understanding of how AMPs function on a molecular level will directly affect the development of antimicrobial drugs in the pharmaceutical industry. Research on AMPs has yet to be expanded from discovery and simple characterization, to an in-depth study using biophysical methods. This will provide the detail necessary for design and development of AMP-based drugs. Pharmaceutical companies who specialise in developing small molecule compound (SMC) antimicrobial drugs are not currently investing in AMP development. This is because insufficient knowledge of AMP mechanisms of action combined with the economic risk of drug development means that this class of antibiotics is only attractive after successful completion of clinical testing. Increased knowledge in the field of AMPs and similar biomolecules will make a fast and inexpensive mechano-structural drug-development approach possible. With further study of their mechanism, AMP-based drugs may have a market potential equal to that of the SMC-based antibiotics currently in use. Development of the experimental techniques proposed for this project may provide a useful high throughput method to investigate the membrane activity of antibiotic compounds. Since AMPs are part of the natural immune response of the body, AMP-based drugs are expected to result in faster recovery and are thought to induce fewer side effects. AMP-based alternative antibiotics will allow clinicians to be able to treat multidrug resistant bacteria, where infections may have lethal outcome at present. Therefore the investigation of AMPs has major implications for the improvement of public health. What will be done to ensure that they have the opportunity to benefit from this research? Research results of note from this project will be disseminated by publication in peer-reviewed scientific journals and to the general public with the help of the Press and Information office at the University of Oxford. Isis innovation (www.isis-innovation.com) helps Oxford University researchers to commercialise intellectual property arising from their research. They work with University researchers on identifying, protecting and marketing technologies through licensing, spin-out company formation, consulting and material sales. Isis funds patent applications and legal costs, negotiates exploitation and spin-out company agreements, and identifies and manages consultancy opportunities.