• To combine our expertise to move from bioinformatic/genetic analysis to the production of novel synthetic antimicrobial molecules, which have had both their structure and activity characterised.
• To develop synthetic strategies to make novel anti-microbial peptides inspired by β-defensins (DIPs).
• To determine the aggregation propensities of naturally occurring defensins and how this relates to biological activity.
• To develop global optimisation methods to solve and predict defensin structures and hence determine the location of amino acids that demonstrate high adaptive selection in mammalian evolution.
• To screen DIPs against molecules that represent potential targets on cell surfaces, and to use cogent mutational studies to locate the interaction interfaces.
• To develop synthetic routes using information from structural evaluation .
This proposal brings together a diverse group of scientists with the broad aim of synthesising and characterising new antibiotics. There has been an enormous rise in resistance to synthetic antibiotics as well as in an increase in the virulence of bacteria, - the so called ‘superbugs’. This was not expected less than 60 years since Sir Alexander Flemming discovered penicillin, and it has catalysed a search for novel molecules to fight bacteria.
Humans and other mammals have an in-built way of dealing with incoming bacteria which is known as the immune response. Part of this is performed by a series of small proteins know as defensins. We produce about 40 of these, which are found on the skin, in tears, in snot and other bodily excretions. It is thought that these defensins work by punching a hole in the membranes surrounding bacteria causing them to burst. However, the precise mechanism of how they work it not known. We have analysed evolutionary changes in the amino acid sequences that make up defensins. We know that some amino acids do not change over time, instead they form internal bonds that keep the defensin bundled up in a ball, but others change rapidly, and we think this is to cope with the changes in bacteria. We want to find out how defensins work. What is the purpose of the fast changing and of the conserved amino acids? We need to know what the structures of defensins are and we will evaluate this using molecular modelling, mass spectrometry and NMR. We need to know what effect changing the amino acids has on the activities of these molecules and so we will test them against panels of pathogens, including some of the antibiotic resistant ones.
Part of this research will think about how defensins interrelate. There is some evidence that they form aggregates, before they interact with membranes, in fact we have studied a dimeric defensin which is much more active than a monomer. We will try to understand which parts of a defensin allow it to aggregate and which parts interact with pathogens. We will examine this phenomena using model cellular environments, both in a ‘wet chemical biology method and also with mass spectrometry and NMR.
Which parts of these molecules need to change as bacteria change and which don’t make a difference and why? Answering these questions will help us to understand the structure activity relationship of defensins, and then help us design new molecules which will be better antibiotics, based on the ones we already have.
1. To combine our expertise to move from genetic/bioinfomatic analysis to novel synthetic antimicrobial molecules.
2. To develop synthetic and recombinant strategies to make novel anti-microbial peptides, inspired by the high adaptive immunity of B-defensins.
3. To employ a range of techniques to characterise the structure and activity of these defensin inspired peptides - DIPs. 4. To develop global optimisation methods to solve defensin structures and hence determine computationally, the
locations of amino acids that display high adaptive selection.
5. Use mass spectrometry based methods, including peptide mass mapping, and ion mobility mass spectrometry to determine where disulfide cross links are and what effect their location has on the structure.
6. Evaluate the aggregation propensity of DIPs and how this relates to activity.
7. Develop routes to inform synthesis with information from structural evaluation.
8. To test the antimicrobial properties of the DIPs in vitro and in vivo.
9. Focus on agents that prove active against Cystic Fibrosis-relevant and multi-antibiotic-resistant pathogens. 10. To discover modes of action of defensins to inform the synthesis of DIPs.
11. Study the interaction of DIPS with model membranes, LPSs and other negatively charged molecules found on animal
12. Screen DIPs against molecules which represent potential targets on cell surfaces and to use cogent mutational studies to locate the interaction interfaces.
13. To develop transferable techniques that may be applied to other systems. 14. Develop the expertise of the individual members of the collaboration.