Tuning a polar molecule for selective cytoplasmic delivery by a pH (Low) insertion peptide

Drug molecules are typically hydrophobic and small in order to traverse membranes to reach cytoplasmic targets, but we have discovered that more polar molecules can be delivered across membranes using water-soluble, moderately hydrophobic membrane peptides of the pHLIP (pH low insertion peptide) family. Delivery of polar cargo molecules could expand the chemical landscape for pharmacological agents that have useful activity but are too polar by normal drug criteria. The spontaneous insertion and folding of the pHLIP peptide across a lipid bilayer seeks a free energy minimum, and insertion is accompanied by a release of energy that can be used to translocate cell-impermeable cargo molecules. In this study, we report our first attempt to tune the hydrophobicity of a polar cargo, phallacidin, in a systematic manner. We present the design, synthesis, and characterization of three phallacidin cargoes, where the hydrophobicity of the cargo was tuned by the attachment of diamines of various lengths of hydrophobic chains. The phallacidin cargoes were conjugated to pHLIP and shown to selectively inhibit the proliferation of cancer cells in a concentration-dependent manner at low pH.