Purdue University Graduate School
2022.05.19 Thomas Dietsche Dissertation .pdf (5.75 MB)

Hydrophobic Modifications to Enhance a Cell Penetrating Antimicrobial Peptide

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posted on 2022-05-19, 16:18 authored by Thomas Allen DietscheThomas Allen Dietsche

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. 


Degree Type

  • Doctor of Philosophy


  • Chemistry

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Jean Chmielewski

Additional Committee Member 2

Herman Sintim

Additional Committee Member 3

Betsy Parkinson

Additional Committee Member 4

Robert Stahelin

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