Developing releasable antimicrobial peptide-polyethylene glycol conjugates by targeting infection site-associated host matrix metalloproteinases

The rapid generation of multidrug-resistant (MDR) bacteria has caused bacterial infections to become a global health concern. Antimicrobial peptides (AMPs), or host defence peptides (HDPs), offer a viable solution to these pathogens due to their broad-spectrum activity and low generation of resistance. In addition, many AMPs possess immunomodulatory properties (e.g., anti-inflammatory activity) that may provide a more robust treatment of infection. However, high toxicity and short biological half-lives have greatly limited the production of clinically available AMP therapeutics. Conjugation of the peptides to delivery vehicles such as polyethylene glycol (PEG) has significantly improved these properties but has also been associated with large reductions in antimicrobial activity, making formulation challenging. In this thesis, an enzymatically releasable PEG-AMP delivery system was developed by incorporating a cleavage sequence susceptible to matrix metalloproteinases (MMPs), enzymes released by the host during the inflammatory response to infection, onto an aurein 2.2-derived AMP. N- vs. C-terminal addition of the sequence found the former to best maintain the activity of the AMP after MMP cleavage, likely due to the maintenance of its amidated C-terminus and higher positive charge. Subsequent conjugation of the cleavable AMP to 2 kDa PEG significantly improved the AMP’s blood biocompatibility in vitro but also eliminated its activity until cleaved by isolated human MMP. This activity was mimicked in an in vivo abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, where both free peptide and conjugate displayed strong activity confirmed to be dependent on the accumulation of MMPs at the infection site, as non-cleavable D-isomeric counterparts of the compounds showed no activity. Following this, the system was expanded to larger PEG molecules by incorporating a tetraglycine spacer between carrier and MMP cleavage sequence. This spacer enabled cleavage of the AMP when bound to 5, 10, and 22 kDa PEG, not possible for the initial peptide, allowing for further improvements in biocompatibility compared to the 2 kDa PEG-AMP conjugate. Altogether, the enzyme-releasable delivery system developed here may provide a suitable platform for the development of infection site-targeting AMP therapeutics where both high biocompatibility and activity can be achieved.Science, Faculty ofChemistry, Department ofGraduat