Using Peptidomimetics to Dissect Activator-Coactivator Protein-Protein Interactions

Protein-protein interactions (PPIs) play significant roles in all cellular activities. The interactions are guided by cellular signals which direct how and where these proteins assemble. The information encoded by DNA is transcribed, for example, as a result of complex PPI networks formed by dynamic transcriptional proteins. If any of the PPIs malfunction, then the resulting dysregulation leads to human diseases such as cancer, neurodegenerative disorders, developmental disorders, and abnormal metabolic activities. At the core of the transcriptional PPI network are proteins known as transcriptional activators that bind to specific sequences of DNA. The other basic component of activators is the transcriptional activator domain (TAD), which functions to recruit coactivators in the process of assembling the transcription machinery. Coactivators play a key role in transcriptional regulation as they serve to interact with multiple protein partners, including activators. The complexes formed between activators and coactivators have modest affinity and are often transient in nature, since they are formed as part of the assembly/disassembly part of the transcriptional machine. Additionally, coactivators use binding sites with large surface areas to form PPIs with activators and other binding partners. This aids the recruitment of the RNA polymerase following the interactions of these classes of proteins. Typically, small molecules tend to be most potent when they act as ligand for proteins such as enzymes that have deeper grooves and small surface areas. However, coactivator proteins have large surface areas and produce moderate affinities when interacting with binding partners. Because of these characteristics, it has been challenging using small molecules to target these interactions. In the case of the target coactivator for this study, Med25, the domain that interacts with activators has two 900 Å2 binding surfaces that are intractable for small molecule ligands. Med25 is dysregulated in many human diseases and there is thus a strong need to identify synthetic modulators that would be useful for mechanistic studies. Towards this end, we first identified minimal binding sequences from known transcriptional activators that interact with the Activator Interaction Domain (AcID). The information gleaned from these studies presented the opportunity to develop synthetic ligands of Med25 AcID using peptidomimetics based on those sequences. Peptoids were chosen as the scaffold due to their ability to resist proteolytic degradation, cover large surface areas and present diverse functional groups as sidechains. We demonstrated that we could design peptoids based on identified minimal peptides that interact with Med25 AcID to recapitulate binding to the Med25. After optimization, the best peptoid exhibited low micromolar affinity for Med25 AcID and good selectivity against a related coactivator. The work yielded promising results leading us to improve the compounds so that that they have intercellular applications. Cell penetrating peptides were appended to the optimal peptoid and the cell permeability assessed by visualizing Vari-068 cells, a triple-negative breast cancer cell line, treated with fluorescently-labeled variants of the peptoid. One version could be seen throughout the cell, including the nucleus, after 6 hours. Taken together, this work demonstrates that we can design mimics of native TADs to interact with larger surface areas with moderate affinities for the target proteins and eventually modify these for efficient intracellular delivery and inhibition.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/169790/1/sdkevon_1.pd