The Design, Synthesis, and Investigation of Functional Cationic Amphiphilic Polyproline Helices (CAPHs)
Antimicrobial peptides are a class of antibiotics that generally act by targeting the microbial cell membrane, resulting in cell lysis. We have designed and synthesized unnatural proline-rich peptides that have been evaluated for antibacterial activity. P14LRRFl is a non-membrane lytic, broad spectrum antimicrobial with efficient mammalian cell penetration. Herein, we report on the elucidation of the mechanism of bacterial action of this peptide. By using the biotin labeled analogue BP14LRRFl, we have isolated protein targets from Staphylococcus aureus and Escherichia coli. Proteomics analysis has identified enolase, a 47 kDa protein, as the intracellular target of P14LRRFl for S. aureus and glycerol dehydrogenase, a 42 kDa protein, for E. coli. Validity of enolase as an intracellular target for the P14LRRAc was confirmed using inhibition kinetics. It was also demonstrated that P14LRRAc binds uncompetitively to enolase. Finally, enolase was also pulled-down from other gram positive strains of bacteria incubated with BP14LRRFl. The therapeutic value of aminoglycoside antibiotics against intracellular bacteria is limited due to their inability to traverse the eukaryotic membrane. While P14LRRFl displays a good reduction in intracellular bacteria, more potent agents are needed to effectively eradicate pathogenic bacteria from mammalian cells. To address this problem, a cleavable conjugate of the antibiotic kanamycin and P14LRR, was prepared. This approach would allow kanamycin to enter mammalian cells as an antimicrobial peptide conjugate, P14KanS, linked via cleavable tether. Potent antimicrobial activity of the P14KanS conjugate was demonstrated with Gram positive and negative bacteria in vitro. Successful clearance of Mycobacterium tuberculosis and smegmatis within macrophages was observed with the antibiotic conjugate, with 93 and 95% reduction, respectively. Additionally, Salmonella enteritidis and Acinetobacter baumannii levels were also significantly reduced in an in vivo Caenorhabditis elegans model as compared to the individual antibiotics, with 70 and 100% reduction, respectively.
Chmielewski, Purdue University.
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