Developing a genetic tag for in vivo protein regulation using PROTACs with companion PET imaging

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Project Summary/Abstract The following proposal describes my aims to use a known protein-ligand pair as a scaffold to create a molecular tool kit composed of small molecule degraders for protein regulation and nuclear imaging probes. Specifically, this molecular tool kit is comprised of a small protein domain, E. coli dihydrofolate reductase (eDHFR), and its small molecule inhibitor, trimethoprim (TMP). TMP can be modified to function as a proteolysis targeted chimeric (PROTAC) molecule capable of protein regulation in a dose-dependent and reversible fashion. Additionally, our group has developed eDHFR as a positron emission tomography (PET) imaging reporter protein capable of imaging engineered cells in vivo. Taken together, my approach would give researchers the ability to modulate protein activity of any desired gene and quantitatively study the phenotypic outputs of these changes in animals and potentially in human patients. PROTAC regulation is the leading ligand-mediated knockdown approach for controlling cellular protein activity. Ligand-mediated strategies allow researchers to tune knockdown intensity with temporal precision. I have demonstrated that the eDHFR-TMP PROTAC system can induce 95% degradation of eDHFR-tagged protein in OVCAR8 cells at nanomolar concentration within 24 hours. I have also demonstrated that this process is reversible and proceeds through a proteolysis-mediated degradation mechanism. We are expanding this technology to determine if we can regulate diverse proteins in multiple subcellular compartments. Ultimately, this suite of molecular tools will provide a modular system for rapidly inducible and reversible knockdown of proteins of interest that can be analyzed quantitatively in clinically relevant models and potentially in human therapies. To advance these molecular tools for in vivo use, I will establish the kinetics of target degradation of the eDHFR-TMP PROTAC in OVCAR8 xenograft tumor models in mice using bioluminescence imaging. I hypothesize that the protein level response of eDHFR-luciferase will lag behind target-occupancy of TMP PROTAC in vivo. Therefore, simultaneous PROTAC administration and PET imaging of eDHFR with established [18F]fluoropropyl-trimethoprim ([18F]FPTMP) radiotracer in mice will accurately quantify PROTAC specificity and kinetic activity. Overall, this work is a novel application of PROTAC and PET technology, where together, these tools can be used to probe expression in vivo, modulate protein activity, and potentially, be incorporated into human immunotherapies to improve therapeutic outputs.