| Proteins and peptides that traverse biological membranes and modulate intracellular signaling networks catalyze scientific discoveries and alter the course of human pathologies. Over 60 polypeptides have passed clinical trials, with 128 currently in the pipeline. In vitro, peptide-based biologics have been designed to directly regulate protein·biomolecule interactions that are pertinent to disease and considered undruggable by small molecule approaches. However in living systems, most fail to bypass the plasma membrane, and hence, the majority of peptide therapeutics modulate cell surface receptors, while few have intracellular targets. This presents a major hurdle for drug design, because a myriad of disease-relevant protein networks reside within the cell interior and are challenging to regulate with traditional small molecules. To alter progression of such pathologies, polypeptides must be designed to efficiently engage the cell interior.;Recently, select classes of natural proteins, synthetic peptides and nonpeptidic polymers have been discovered to bypass cellular membranes and deliver medicinal cargo into the cytosol. Herein, we enhance the potential of biological therapeutics through exploring the physicochemical compositions and membrane trafficking processes that allow proteins and peptides to access the cell interior. We have developed cell-based systems that provide information on intracellular levels attained by exogenously supplemented peptides and have made use of our assays to discover that small, folded proteins containing five arginines arrayed precisely about three contiguous faces of an a-helix enter into living cells via endocytosis, release from early endosomes characterized by the guanosine triphosphatase Rab5, and achieve intracellular concentrations that dwarf those of the most commonly applied cell-permeable peptides . Additionally, we have furthered our understanding of how cells regulate the partitioning of endocytosed proteins into the cytosol through genome-wide RNAi screening at Yale's High-Throughput Center for Cell Biology. Our screen has allowed us to identify unprecedented candidate knockdowns that modulate the ability of polypeptides to enter cells and release from vesicles of the endocytic system. We anticipate that the methodologies and discoveries described herein will catalyze the identification of physicochemical peptide features and cellular trafficking pathways that allow biologics to efficiently reach the cell interior and thereby guide fulfillment to the promise of peptides as ligands for the unclruggable proteome. |
