Hydrophobic Modifications to Enhance a Cell Penetrating Antimicrobial Peptide
The rapid emergence of antimicrobial resistance has created a daunting challenge in which many current antibiotic treatments have been rendered useless. Furthermore, pathogens such as Shigella and Listeria have evolved to hijack host mammalian cells, providing them a safe haven from poorly permeable antimicrobial drugs. This looming threat is juxtaposed by the lack of new FDA approved antibiotics as well as emerging strains that are resistant to drugs of last resort, such as vancomycin. Therefore, it is of the utmost importance to develop cell-penetrating antibiotic therapies with novel mechanisms of action.
To combat this antimicrobial epidemic, the Chmielewski group has combined valuable characteristics of antimicrobial peptides and cell-penetrating peptides (CPPs) to develop a class of synthetic peptides composed of a cationic amphiphilic polyproline helix (CAPH) scaffold. This scaffold contains both hydrophobic and hydrophilic residues to grant the molecule amphiphilic characteristics. To improve activity, we have demonstrated that functionalizing CAPHs with hydrophobic moieties at the N-terminus improved cell uptake and, in some cases, antibacterial activity. These N-terminal modifications are installed in a facile manner on resin, making them easily adaptable to other peptides to improve their cell penetration or activity against intracellular pathogenic bacteria. By altering CAPHs to bear the 5-carbon aliphatic chain, we found that Pentyl-P14 was effective at clearing the intracellular pathogen Shigella flexneri.
In addition to N-terminal CAPH analogues, we have also modified the cationic amphiphilic polyproline helix (CAPH) scaffold with rigidly-placed hydrophobic functionalities, thereby enhancing the cell-penetration and antimicrobial activity of these compounds. One CAPH, P14- Pentane, was found to clear intracellular MRSA within macrophage cells. Furthermore, through mechanistic studies with large unilamellar vesicles (LUVs), we provide evidence that the hydrophobic modifications can increase the propensity of the CAPHs to interact with the phospholipid bilayer, providing an explanation for the observed increased cell uptake.
After establishing the potency of N-terminal and sidechain hydrophobic modifications in the CAPH skeleton, we synthesized a small library of analogues that incorporated a combination of 20 these two modifications, as well as a shorter, more rigid cationic residue which has also shown the ability to improve cell penetration in CPPs. One promising CAPH derivative, CAPH-4, was identified as it showed an increase in cell uptake of over 190-fold as compared to P14LRR in HeLa cells. Furthermore, this derivative was found to translocate across the mammalian cell membrane via a direct transport mechanism even at a sub-micromolar concentration. Overall, modifying the CAPH scaffold with different hydrophobic moieties has proven to be a powerful strategy to improve cell penetration and antimicrobial activity. This is crucial for addressing the need of novel antibiotics that can target and eradicate intracellular bacteria in the coming era of drug-resistant bacteria.
- Doctor of Philosophy
- West Lafayette