DE NOVO CATIONIC ANTIMICROBIAL PEPTIDES AS THERAPEUTICS AGAINST PSEUDOMONAS AERUGINOSA
Cationic antimicrobial peptides (CAPs) are a very diverse group of amphipathic agents that demonstrate broad activity against Gram-positive and -negative bacteria. To overcome the obstacle of drug resistance among bacterial pathogens, CAPs have been extensively investigated as a potential source of new antimicrobials with novel mechanisms of action that may complement current antibiotic regimens. However, the suppression of antimicrobial activity in biological conditions (e.g., physiological salt concentrations, serum) constitutes a major challenge to the successful development of CAPs for clinical applications. We hypothesized that CAPs with optimized amphipathic structures can be designed de novo to enhance antibacterial activity and selective toxicity in environments that are generally challenging to host-derived peptides. Furthermore, the antibacterial efficacy will positively correlate with length, charge, Trp content, and helicity.
Three specific aims were developed to address this hypothesis. The first aim addressed the design of amphipathic peptides to evaluate the influence of helicity, length, and Trp content on activity. Using a base unit peptide approach, we synthesized a multimeric series of 12-residue lytic base unit (LBU) composed of Arg and Val residues, positioned to form idealized amphipathic Ñ-helices. Another series of LBU derivatives (WLBU) was engineered by substituting Trp residues on the hydrophobic face. The correlation between length and helicity was established by circular dichroism analysis. Bacterial killing assays revealed no appreciable increase in activity for peptides longer than 24 residues. In addition, the inclusion of Trp residues in the hydrophobic face increased potency and selectivity in a novel co-culture system utilizing bacteria and primary cell lines, which led to the selection of WLBU2 as the shortest peptide (24 residues in length) with the highest potency. The second objective was to examine the selective toxicity of WLBU2 in biological or biologically-derived media. In contrast to the human CAP LL37, the peptide WLBU2 displayed potent activity against Pseudomonas aeruginosa in the presence of human serum and human blood ex-vivo, with no detectable red blood cell lysis or toxicity to human monocytes at all test concentrations. Finally, we demonstrated potent activity of WLBU2 in intraperitoneal and intravenous mouse models of Pseudomonas aeruginosa infection. WLBU2 not only protected mice prophylactically but also eradicated P. aeruginosa from the blood and other tissues at 3 to 4mg/kg. Furthermore, WLBU2 displayed only a minor stimulatory effect on inflammatory cytokines, notably IL1-£] and TNF-Ñ. Consistent with our in vitro studies, the in vivo data provide strong evidence for the potential application of WLBU2 in the treatment of systemic infection due to P. aeruginosa.
Advisor:Bruce A. McClane; Michael Cascio; Michael A. Parniak; Sharon L. Hillier; Timothy A. Mietzner; Ronald C. Montelaro
School:University of Pittsburgh
School Location:USA - Pennsylvania
Source Type:Master's Thesis
Keywords:molecular virology and microbiology
Date of Publication:01/11/2006