Design of Protein-Degrading Peptide Bioconjugates

Non-technical description The classification of a drug substance as a medicine or poison depends on several factors including the amount given, how rapidly it is given, where it is given, and drug specificity. This proposal focuses on the latter, that is, enabling the drug to go directly to a desired location so that it can specifically kill the cells that are causing a specific disease. The drug in this case is a tiny protein called a peptide, akin to a short, beaded necklace where each bead is an amino acid. These peptides can be designed, by stringing the beads together in a specific order, to delete bad disease-causing proteins in cells. However, peptides are not specific and are not terribly efficient at getting inside cells. One way to improve their specificity is to attach the peptide to a large targeting protein that transports the drug to the site where it needs to go. To improve their cell entry, another short polymer strand is attached that allows the peptide to slip inside the cell when it gets there. While this sounds like a good plan, the only way it works is if the peptide can detach from the targeting protein at the right time. If it detaches too early before it reaches the cell where it needs to be, it might go to undesired locations and lead to toxic or poisonous effects. If it detaches too slowly, it will be ineffective. This NSF proposal will study how to attach peptide drugs to the targeting protein and how the speed of detachment affects the ability of peptide to exert its functional effect. Studying the mode of attachment and speed of release will be beneficial for scientific progress and will have a significant impact in several biotechnology fields such as antibody-drug conjugates, peptide-conjugation, and peptide delivery, where effective delivery strategies can be used to build better drugs and advance national health. Furthermore, educational and outreach activities will be integrated throughout this research through a common theme focused on promoting peer-to-peer learning and empowering young aspiring scientists to take up leadership positions in communicating STEM ideas to the broader public. Education and outreach goals will include the development of interactive modules for the CATALYST Academy workshop and the 4H Focus for Teens Program. Technical description This NSF project will develop a new class of bioconjugates to improve the intracellular delivery of Peptide-based proteolysis TArgeting Chimeras (PepTACs) toward their use in proteome-editing applications. PepTACs are bispecific peptide-based ligands that induce targeted protein degradation via the ubiquitin proteosome system. By employing a peptide ligand that recruits an E3 ubiquitin ligase and another that targets a protein of interest (POI), PepTACs facilitate the formation of a ternary complex, POI ubiquitination, and subsequently rapid catalytic POI degradation by the proteasome. However, these heterobifunctional peptide-ligands are not cell-specific and their potency is limited due to inefficient intracellular delivery. Building on the benefits of peptide-based ligands and the clinical success of antibody-drug conjugates, this NSF project aims to improve cell-specific PepTAC delivery using antibody carriers and cell-penetrating oligothioetheramides (CPOTs) for efficient cytosolic delivery. The resulting optimized product, an antibody-PepTAC conjugate (APC), will be designed to enable rapid and efficient intracellular protein degradation mediated by PepTACs. The proposal outlines several experiments to study the impact of conjugation site on APC transport and the release kinetics of PepTAC-CPOT constructs from the antibody within the endosomal compartment. Parameters from these studies will be correlated to APC intracellular transport and protein degradation. By studying these critical design features, this project will lay the groundwork for the effective delivery of PepTACs, unlocking their vast potential as invaluable tools for cell-specific targeted protein degradation. The ability to rapidly engineer and deliver high-affinity protein degraders against any intracellular target will be of great significance to experimental chemical biology researchers seeking to decipher protein interactions and unravel biological networks. This project is particularly impactful as a biological tool, providing an alternative method for achieving protein degradation (knockout) without the need for genetic materials or alterations to the cells genetic content. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.