Cytosolic Delivery of Proteins

Abstract

In medicine, current methods to target intracellular proteins have largely been limited to small molecule drugs as they can freely diffuse across the plasma membrane. However, their lack of specificity and inability to target large protein-protein interfaces are limitations. Proteins, such as antibodies, overcome these limitations but they are too large to diffuse into the cell. Intracellular targets such as RAS and MYC are therefore deemed “ungruggable”. A method to deliver proteins into live cells in their intact form could therefore prove very useful in research and medicine. We present work done on pHD108, a pH-sensitive pore-forming peptide. Through libraries and screens and synthetic molecular evolution, we imparted the parent peptide melittin with the ability to be potent (activity at 1:1000 peptide to lipid ratio), triggered by low-pH only (pH < 6), and form macromolecule-sized pores (∼8.2 nm radii in POPC bilayers). We sought to harness these unique properties for protein delivery into cells. Since endosomes become acidic (pH ∼6), we hypothesized that cells endocytosing protein drug as well as pHD108 peptide will allow the peptide to become active and form pores in the endosome, thus releasing the protein drug into the cytosol. We show evidence supporting this through a novel apoptosis assay based on P. aueruginosa exotoxin a domain III (PE-III, 37 kDA). PE-III is only cytotoxic when delivered into the cytoplasm else it gets degraded in the lysosome. With D-pHD108 treatment, we see apoptosis, indicating endosomolytic activity. We also see that pores in cells are large enough for IgGs to pass through. Modifications to the peptide has revealed an importance in membrane binding as anchoring to membranes through an acyl tail can increase activity. Together, this shows pHD108 is functional for this application but a roadmap for further optimizations should be followed as well.