Nanodiscs as high-resolution structural models for native like antimicrobial peptide-membrane interactions

It is now well accepted that the rise of antibiotic resistance is one of the biggest global crises the world will face. If we do not take comprehensive measures, the consequences will be terrible, both on a human and financial level. More people will die from infections than from cancer, and the world will have its overall economic output reduced by 100 trillion dollars. It is obvious that solving a problem of this magnitude and complexity will require a global effort, and one of the partial solutions will be to ensure that we will still have access to effective antibiotics. Antimicrobial peptides are a promising class of molecules for the development of future antibiotics, but it has so far proved difficult to achieve in practice. An important reason for this is our inability to understand how antimicrobial peptides affect the bacteria at an atomic level. In this project, we have adopted the very latest and best model system for bacterial membranes, nanodiscs, to gain a detailed insight into how the peptides kill bacteria. During the project's last year, we have established the production of nanodiscs, and started the painstaking work of studying how the peptides destroy membranes. The work with the nanodiscs has now enabled us to create an established technique that has been published so that other research groups can use it in their research. In addition to the nanodiscs, we have also developed NMR-based models that quantitatively measure trans-membrane transport of peptides, and of the peptides' ability to influence membrane transport of ions and small molecules. In this area, the project has come a long way, and the inventors of the technique are in the process of patenting and commercializing this technique. During the project, we have also established a collaboration with an internationally leading research group at the University of Groningen, the Netherlands, which works with theoretical modeling and atomic force microscopy (AFM) of the peptides we investigate in nanoAMP. In order to put this knowledge into a further context the project has been incorporated into the DigiBiotics consortium, and the new knowledge gives us new opportunities to design new peptides that can be developed into medicines for the benefit of future generations. This work has led to a joint publication that has been submitted to a leading journal. Through the DigiBiotics consortium, the project has characterized a marine natural product that facilitates ion transport across the membrane and in this way is a natural mimic of antimicrobial peptides. NanoAMP has been an important contributor to the DigiBiotics consortium, and has contributed to establishing and verifying new methods for investigating membrane interactions. These contributions are incorporated into the research pipeline that has been established by DigiBiotics.

My assessment is that the impact of nanoAMP has been significant and further enlarged by the integration with DigiBiotics. Scientifically, the impact can be (partly) seen in the scientific publications originating from the project, here detailed in the next capter, but more importantly, nanoAMP has attracted interest from stakeholders outside the project. An industry-PhD has used the knowledge on optimal design of cyclic antimicrobial peptides to investigate the concept of creating anti-colonizing surfaces using surface linked cyclic hexapeptides to confirm proof-of-principle investigation on covalent attachment of antimicrobial peptides.

Antimicrobial peptides are also one of the very few truly novel antimicrobial molecules discovered the last 30 years, and may be an important tool to avoid the emerging antibiotic crisis. The peptides are antibiotic through a unique biophysical mode of action where they lyse (dissolve) the bacterial cell membrane. The key interaction in any of these mechanisms is the interaction between the peptide and the cell membrane, and several models of the mode of action has been proposed, and in all a key step involve embedding of the peptide in the lipid bilayer. The detailed structure of the peptide in the lipid bilayer of bacterial cell membrane has until now eluded us, due to insufficient experimental technology. Recently, Nanodiscs, a novel membrane bilayer experimental model, has been developed for the study of membrane bound proteins. The Nanodiscs are round, stable, uniformly nano-sized patches of lipid bilayers that can give rise to high resolution NMR experiements. In the present proposal we propose to create Nanodiscs with lipid compositions resembling several important cell membranes, from bacteria, cancer cells, normal cells and mitochondria. We will then design and synthesise specifically labelled probe peptides to verify our ability to obtain not only high resolution structures of the peptides, but also to both qualitatively and quantitatively assess the membrane perturbation caused by these peptides. We will also design libraries of antimicrobial peptides to derive quantitative structure activity relationship towards different cell membranes thereby for the first time enable us to rationally design antimicrobial peptides with preselected activity, selectivity and other properties important for the development of antimicrobial peptides to new antibiotic drugs. Finally we will study the intriguing application of antimicrobial peptides as immunogenic anti-cancer agents.