Potential of Host Defense Peptide Prodrugs as Neutrophil Elastase-Dependent Anti-Infective Agents for Cystic Fibrosis

Abstract

Host defense peptides (HDPs) are short antimicrobial peptides of the innate immune system. Deficiencies in HDPs contribute to enhanced susceptibility to infections, e.g., in cystic fibrosis (CF). Exogenous HDPs can compensate for these deficiencies, but their development as antimicrobials is limited by cytotoxicity. Three HDP prodrugs were designed so their net positive charge is masked by a promoiety containing a substrate for the enzyme neutrophil elastase (NE). This approach can confine activation to sites with high NE levels. Enzyme-labile peptides were synthesized, and their activation was investigated using purified NE. Susceptibilities of Pseudomonas aeruginosa to parent and prodrug peptides in the presence and absence of NE-rich CF human bronchoalveolar lavage (BAL) fluid and different NaCl concentrations were compared. The effect of the HDP promoiety on cytotoxicity was determined with cystic fibrosis bronchial epithelial (CFBE41o-) cells. NE in CF BAL fluids activated the HDP prodrugs, restoring bactericidal activity against reference and clinical isolates of P. aeruginosa. However, activation also required the addition of 300 mM NaCl. Under these conditions, the bactericidal activity levels of the HDP prodrugs differed, with pro-P18 demonstrating the greatest activity (90% to 100% of that of the parent, P18, at 6.25 g/ml). Cytotoxic effects on CFBE41o-cells were reduced by the addition of the promoiety to HDPs. We demonstrate here for the first time the selective activation of novel HDP prodrugs by a host disease-associated enzyme at in vivo concentrations of the CF lung. This approach may lead to the development of novel therapeutic agents with low toxicity that are active under the challenging conditions of the CF lung.

Antimicrobial and Biophysical Properties of Surfactant Supplemented with an Antimicrobial Peptide for Treatment of Bacterial Pneumonia

“…Whereas the main focus of our in vitro experiments was the maintenance of surfactant and antimicrobial activities of potential AMP-based surfactant preparations, it should be noted that it has also been reported that AMP can have cytotoxic effects toward mammalian cells (10,26,33). Such effects could potentially limit the therapeutic applicability of this approach.…”

“…To accomplish this goal, surfactant adsorbs and spreads rapidly to form a surface film consisting of lipid-condensed and lipid-expanded regions at the air-liquid interface, which upon lateral compression forms a stable multilayer structure capable of reducing surface tension values to near 0 mN/m (10). Through its ability to spread throughout the lung and open up collapsed lung areas, exogenous surfactant therapy has been shown to reduce mortality of premature infants afflicted with neonatal respiratory distress syndrome (10,11).…”

Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis

“…Both protocols for collection were approved by the Beaumont Hospital Research (Ethics) Committee. The NE content was determined by measuring the cleavage of N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as described previously (16).…”

“…The colocalization of P. aeruginosa and NE allows the potential cytotoxic effects of the AMPs (pro-HB43 and pro-P18) to be confined to the site of infection (16). However, a significant scope to improve the selectivity of pro-AMPs remained.…”

“…One potential source of new anti-infectives for CF is antimicrobial peptides (AMPs) (15,16). These short amphipathic peptides, composed of hydrophobic and charged amino acids, are crucial components of the innate immune system.…”