ENGINEERING SEQUENCE-DEFINED OLIGOTHIOETHERAMIDES FOR EFFECTIVE INTRACELLULAR DELIVERY

Abstract

The discovery of cell-penetrating peptides (CPPs) over three decades ago uncovered a novel method for transporting various cargoes into cells. Although promising, CPPs have several drawbacks such as rapid metabolic degradation by proteases, immunological response, undesired interactions with the biological milieu, and nephrotoxicity. Recently, the Alabi group developed the rapid assembly of sequence-defined oligothioetheramides (oligoTEAs). OligoTEAs have three distinct advantages over native peptides, including i) abiotic design to reduce proteolytic degradation and immune response, ii) backbone and pendant group access to tune interactions, and iii) diverse monomers for massive compositional space. Previous studies indicate that a combination of cationic and hydrophobic residues is critical for translocation across cellular membrane. Thus, we assembled an 8-residue oligoTEA library composed of a hydrophilic backbone, cationic monomer, and hydrophobic monomer. As discussed in chapter 2, by probing this initial library we found that four or less cationic residues is insufficient for efficient uptake of oligoTEAs, and hydrophobicity plays an important role in their uptake. Further studies reveal the discovery of non-charged cell-penetrating oligoTEAs (CPOTs) that undergo efficient cellular uptake with low cytotoxicity, and outperforms R9, a widely-used CPP. Chapter 3 details the sequence-controlled assembly of these CPOTs and their uptake efficiency across various cell lines. Studies of different cell entry mechanisms of the best CPOT performer suggests that its primary mode of uptake occurs via direct membrane translocation. Moreover, the guanidine headgroup also appears to be a crucial chemical functionality for bactericidal activity. In fact, many antimicrobial peptides (AMPs) have similar chemical residues to CPPs, with the one important exception that they have a larger percentage of hydrophobic groups. Hence, we examined the effect of different cationic pendant groups and backbone hydrophobicity on the antibacterial activity of oligoTEAs in chapter 4. CPOTs show strong promise for the intracellular delivery of therapeutics. As such, we investigated their use in the delivery of vancomycin, which has limited activity against intracellular bacteria due to its impermeability to host cells. In chapter 5, we present the assembly of reducible and non-reducible vancomycin-CPOT conjugates and their efficient transport into multiple mammalian cells towards the treatment of intracellular pathogens.