Neutrophil elastase-activated host defence peptide prodrugs kill pseudomonas aeruginosa in a cystic fibrosis lung milieu

Background: Host defence peptides (HDPs) are short antimicrobial components of the innate immune system. In cystic fibrosis (CF), deficiencies in HDPs rationalises exogenous application but cytotoxicity limits development as therapeutics. Previously, neutrophil elastase (NE)-labile HDP prodrugs were designed with a promoiety successfully reducing cytotoxicity (1). However, remaining issues with selectivity and cytotoxicity require the optimisation of the promoiety and peptide sequence. This study further refines design. Methods: Lability to purified NE was confirmed with HPLC and MALDI-TOF MS. New HDPs were incorporated into the prodrug model. The susceptibility of Pseudomonas aeruginosa to the peptides was compared with/without NE-rich CF bronchoalveolar lavage (BAL) fluid. The effect of the promoiety on cytotoxicity was determined with CF bronchial epithelial (CFBE41o-) cells. Results: The use of new peptide sequences yielded the required improvements. One pro-HDP, pro-WMR, maintained low antimicrobial activity in the absence of NE, performing better than the previous pro-HDPs, with a bactericidal activity at 25μg/ml that increased from 8.4% to 91.5% with the addition of 25%v/v BAL (p=0.0008). Selectivity was also greatly improved (IC 50 against CFBE cells ≥300μM). Conclusions: Selective activation by a host disease-associated enzyme at physiological concentrations was demonstrated here and the promoiety successfully reduced cytotoxicity. The study has produced pro-HDPs, with greatly reduced host toxicity, that function in the challenging conditions of the CF lung. 1. Forde et al. Antimicrobial agents and chemotherapy. 2014;58(2):978-85.