The Location of the Hydrophobic Proteins SP-B and SP-C in Fluid-Phase Bilayers

Abstract

Targeting protein-protein interactions (PPI) within the cell is an appealing prospect for cancer research and therapy. Methods to inhibit PPIs have been limited though due to the ineffectiveness of small molecules for this task, and to the inability to get macromolecule cargoes -such as antibodies -into the cytosol. Because of this, targets such as RAS and MYC are sometimes considered ''undruggable'' even though they have been studied for decades. To address this, we are developing pH-sensitive pore-forming peptides to enable endosomal escape of endocytosed protein cargo. One such peptide, pHD108, is a pHtriggered gain-of-function variant of melittin, a membrane permeabilizing peptide found in bee venom. In synthetic unilamellar vesicles, pHD108 has no poration activity at pH 7; at pH % 5.5 it releases macromolecules from lipid vesicles at very low peptide concentration. In this work, we have shown that, with a low pH pulse, pHD108 allows 150 kDa proteins (IgGs) through the plasma membrane. High concentrations of peptide are needed, however, so steps to increase potency has yielded an optimal acylation strategy of a 6-carbon acyl chain at the N-terminus. To assess endosomolytic activity, a novel assay was developed using domain III of Pseudomonas Exotoxin A. When given to cells alone, the exotoxin does not induce apoptosis but when delivered to the cytosol it will induce apoptosis. pHD108 shows activity here. To further increase potency we plan to screen a focused library of peptides with rationally modified sequence variations while employing the optimal acylation strategy. Hits found there will be characterized further. Our findings here will ultimately allow researchers to target intracellular PPIs in living cells using exogenous antibodies.