Multi-mode imaging to underpin a drug development pipeline for a new, peptide class of antibiotics

The 2016 Review on Antimicrobial Resistance (AMR) predicts that, unless action is taken, around 10 million deaths per year will be attributable to AMR by the year 2050. Commissioned by the Wellcome Trust, a recent pipeline portfolio review of alternatives to antibiotics recommends “strong support for funding while monitoring for breakthrough insights regarding systemic therapy” for a tier of approaches that include antimicrobial peptides (AMPs). The review however presents the prevailing wisdom that AMPs are unsuited for systemic administration as they are poorly tolerated in animal models and susceptible to degradation. This substantially limits the scope of infection settings where AMPs can be used and hence their future development.

In a recent structure-activity-relationship study, researchers at KCL and the National Infections Service of Public Health England (soon to form part of the National Institute for Health Protection; NIHP) identified an analogue hit of a naturally occurring AMP that has substantially improved, broad spectrum, antibacterial properties and which has been shown to be effective in mouse models of bacterial lung infection when delivered intravenously (i.v.). We have filed a patent application which covers this analogue and several others. However, the pathway for successful development of this new class of antibiotics is far from clear, with new approaches needed to understand pharmacokinetics (PK), biodistribution and pharmacodynamics (PD), the origin of any toxicity, interactions with the innate immune system and the ability of this class to eradicate intracellular bacteria which are increasingly suspected as being the cause of clinical failure, relapses and resistance after antibiotic therapy.

We propose that a multi-mode imaging approach is the key to addressing these substantial barriers. Specifically, this iCASE studentship, which partners with PHE/NIHP, aims to produce and characterise a panel of differently labelled AMP analogues that will enable in vitro and in vivo fluorescence imaging and in vivo radiopharmaceutical tracing. Year 1: At KCL – the student will be trained in solid-phase peptide synthesis, and will design, prepare and purify a series of fluorescently labelled derivatives of pleurocidin (and its own analogues), as well as the first hexadentate HydrOxyPyridinOne (HOPO) conjugate analogues of pleurocidin. At PHE/NIHP – the student will test the antibacterial potency of these analogues to understand by how much the activity of the AMP is affected by labelling and if it can be tolerated. Year 2-3: At KCL – informed by testing at PHE/NIHP, any differences in mechanism of action, resulting from labelling, can be understood through an established combination of biophysical and metabolomic experiments. If the activity of the labelled analogues is acceptable then (co)localisation fluorescence imaging at the Nikon Imaging Centre will test which AMPs reach intracellular bacteria and better understand any cytotoxicity. At PHE/NIHP the student will screen the new lead AMP analogues of pleurocidin against bacterial transposon libraries and conduct directed evolution experiments to help identify signatures of resistance from bacteria surviving within macrophage or in subsequent in vivo infection models. Years 3-4: At KCL – Alternative labelling strategies (N/C-termini, additional lysine, different HOPO conjugates etc) and further fluorescent labels can be investigated as required, in particular those suited for in vivo biodistribution studies. These will also be screened for activity at PHE/NIHP. Depending on outcomes, near-IR fluorescent and/or 68Ga labelled HOPO—pleurocidin conjugates will be evaluated for use in PK, biodistribution and PD studies.

This iCASE studentship will show that the ability to generate, characterise and evaluate a range of differently labelled AMPs will have broad applicability in defining their in vivo performance and will have a substantial impact on removing some of the barriers to the translation of this hitherto underexploited natural resource.