Development of Cell Permeable PROTACs

The next logical step in the development of the PROTAC technique as a viable therapeutic option was the design of a PROTAC scaffolding that would be independently cell permeable, negating the need for microinjection as the method for PROTAC delivery. A cell permeable PROTAC would also lessen the molecular biological manipulations necessary for PROTAC activity, emphasizing the capacity for the technique as both a tool for understanding protein function in the cell and as a potential therapeutic. 

In order to generate a cell permeable PROTAC, an alternative E3 ligase ligand was required. The IkBcx phosphopeptide functioned successfully to recruit the E3 ligase components, but the negatively charged 

In order to ensure the cell permeability of the new PROTAC design, an eight amino acid polyarginine tag was included on the carboxy-terminus of the E3 ligase-targeting peptide sequence of the PROTAC. This significant positive charge serves to facilitate translocation of the PROTAC into cells via a mechanism that mimics the HIV Tat protein.  

In order to observe the effects of PROTAC-4 on FKBP, a HeLa cell line stably expressing a GFP-tagged FKBP was generated. Thus, loss of the target protein could be visually assessed by diminished fluorescence. The GFP-FKBP cells were then treated with PROTAC-4, which was simply added to the cell 

---