Intracellular protein delivery with the use of endocytosis routing sequences

One of the greatest challenges to overcome in drug development is the efficient translocation of protein-sized drugs into cells, because the mammalian cell membrane acts as a major obstacle to these hydrophilic large molecules, which could otherwise be highly specific, efficient, and tolerable pharmaceuticals. Internalization of these molecules can be achieved by clathrin-independent endocytosis (such as lipid-raft mediated/caveolar endocytosis), and this pathway is exploited by endogenous proteins, bacterial toxins (cholera and tetanus), and viruses (murine polyomavirus and echovirus 1), because this pathway tends to fuse with lysosomes only after a very long endosomal retention time, if at all. This endocytic mechanism is an attractive target to deliver functional proteins without degradation, and the leaky endosomes forming in the process allow direct escape for the molecules before moving to other cellular locations. The surface of these lipid rafts and caveolar pits are composed of various glycosphingolipids, especially of mono-, di-, and trisialotetrahexosylgangliosides (GM1, GD1a, GT1b), which are the major receptors for the natural cargoes. Binding and clustering the gangliosides induce an endocytic mechanism, where lysosomal fusion is negligible, allowing the proteins to reach the cytosol or undergo transcytosis. Many delivery systems fail to avoid lysosomal entrapment, or the molecule responsible for the internalization is required to be used at therapeutically irrelevant, high concentrations. Interpreting the glycan code by studying how gangliosides trigger endocytosis could be the key to solve these problems. Interest in binding gangliosides has already arisen; however, high-affinity molecular recognition is still a great challenge. The specific targeting of ganglioside GM1 is especially sought, because this ganglioside, while normally being expressed in many mammalian cell types, is highly abundant in cancerous cells. Therapeutic protein levels in the extracellular fluid yield 100–500 nM; therefore, a high-affinity interaction is needed to create a cell membrane enrichment that facilitates sufficient material flux in clinical applications. Our main goal was to achieve nanomolar delivery of large proteins (up to the size of antibodies) via lipid raft-mediated endocytosis. We aimed to use a non-toxic peptide tag to mimic the ganglioside-mediated internalization of endogenous and exogenous proteins; therefore, we set out to find a minimal motif that can bind ganglioside GM1 with high affinity and specificity. By focusing on a structurally well-defined receptor and conducting a thorough biophysical characterization of the interaction, we aimed to open a way to structure-based design, which is rare in protein delivery approaches. We set out to investigate the ability of the characterized peptidic tag to deliver large proteins into the cells, while rigorously monitoring its toxicity, mechanism of entry, and the tendency to fuse with lysosomes. Using a medicinal chemistry approach, we set out to establish a structure–activity relationship, to gain insight into the binding mechanism, and to improve the enzymatic stability while retaining high affinity