| Lipidation is a post-translational modification that covalently attaches lipid groups to proteins to restrict their motility to the cell membrane. In this study, we proposed to induce artificial protein-membrane anchorage through the use of a rationally designed Protein-Membrane Anchor (PMA) to inhibit a protein's motility and function within the cell. We hypothesized that induced membrane anchorage of proteins can hold significant therapeutic value when applied to cancer-promoting cell-signaling proteins. Our proof-of-concept PMA 1 was able to sequester and immobilize STAT3, a 93 kDa protein, to the plasma membrane in breast cancer cells. To the best of our knowledge we are the first to design a PMA that targets and restricts the motility of a soluble cytosolic protein. Unfortunately, our second generation PMAs, which incorporated more drug-like, nonphosphorylated molecules, were unable to localize STAT3 to the plasma membrane. However, the PMAs did inhibit STAT3 nuclear translocation in IL6 stimulated breast cancer cells, suggesting that they might be anchoring STAT3 on intracellular membranes. To prevent the physiological drawbacks associated with using large lipidic groups on drugs, we designed prodrug-like PMAs that activate in situ, called PPB-PMAs. The PPB-PMAs incorporate a motif that is recognized by farnesyltransferase to install a farnesyl group, inducing membrane anchorage. Our proof-of-principle PPB was able to undergo farnesylation in an in vitro enzymatic assay as well as anchor in the plasma membrane in breast cancer cells. Currently, the PPB-PMA is being tested for its ability to anchor its target protein, FKBP12. The versatility of the PMA strategy was also demonstrated through the use of metal coordination complex PMAs. These PMAs were able to selectively immobilize phospho-peptides to lipid bilayers. The progress towards designing, synthesizing and testing the novel PMAs is reported herein. |
