Expanding the toolbox for the study of antimicrobial peptides

There is an urgent lack of new antibiotics in the face of an ever-expanding antimicrobial resistance crisis. The fact that fewer new classes of antibiotics are being developed, and resistance soon follows newly available antibiotics, only serves to underline the urgency of the matter. There is a clear need of a paradigm shift with regards to antibiotics, and one such hope is antimicrobial peptides (AMPs). AMPs are an integral part of the innate immune systems of most organisms within the domains of life; since their discovery they have become of significant interest as a new type of antimicrobial agent, due in part to the low capacity of bacteria to develop resistance mechanisms towards them. Despite their potential, and lengthy study so far, establishing the specifics of the mechanism of action of many AMPs remains difficult– particularly of those that target the bacterial cell membrane. This lack of understanding limits the ability to rationally design new AMPs with a view to developing new antimicrobial agents. The aim of this work was to help identify new potential hit compounds through NMR structure elucidation, and to develop new methods that would give greater insight into the activity of membrane active AMPs. This in turn could help enable the rational design of new AMPs. WIND-PVPA, a method to quantify permeabilities of water and ions as a means to evaluate the disruptive capabilities of AMPs, was developed. This was demonstrated on a number of AMPs, and it was shown that WIND-PVPA can identify AMPs that have strong, selective, membrane disruptive activities such as the AMP WRWRWR, as well as more modestly disruptive AMPs such as KP-76. The WIND-PVPA was further used with a non-AMP membrane active natural product – lulworthinone – that was characterised over the course of the project. The findings of the study helped classify lulworthinone as a non-disruptive membrane active agent. In addition, microscale thermophoresis (MST) was shown to be a viable method by which the binding and partition coefficients of Trp-rich AMPs can be determined, and it was shown that the derived lipid-bindings of the AMPs correlates well with their bactericidal activity. Both WIND-PVPA and MST have expanded the toolbox available to the study of AMP-lipid interactions and can be used synergistically to give greater insight into the possible mechanism by which AMPs act, by helping to identify interesting cases, such as non-disruptive AMPs with potent activities. In summary, the methods developed have great potential that can be further refined into robust methods that can greatly assist in the deconvolution of AMP activity and can open up possibilities of the rational design of membrane active AMPs as a new generation of antimicrobial agents.