Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

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Project Abstract The ability to control biomolecules in situ is critical to the experimental process. Proteins are especially important to understand as they are central to cellular function, cell signaling, and living organism processes (e.g. metabolism, tissue development, and immune function). There is an unmet need for a small molecule approach to “tunably” and reversibly regulate expression of an imaging-compatible protein tag to investigate protein function(s). Such a tag would allow multi-modal imaging, including fluorescence and in vivo positron emission tomography (PET), allowing investigators to detect, then control, the tagged protein in cells and animals using small molecule ligands. For example, fluorescence imaging of the tag could guide the proper timing for protein knock-down based on subcellular localization or protein-protein interactions in vitro, and PET imaging of the tag could guide the regulation of proteins modulating cellular trafficking in vivo. A specific example of the in vivo application is in the process of developing chimeric antigen receptor (CAR) T-cell therapies for solid tumors, where understanding in vivo biodistribution, efficacy, and toxicity using imaging would be crucial, and importantly, controlling the cell surface expression of the CAR may have a large impact on that biodistribution, efficacy, and off-tumor toxicity. Chemical derivatives of the small molecule antibiotic trimethoprim (TMP) have been developed into multi-modality imaging probes by our group and others. The objective of this proposal is to set a standard for imaging-compatible protein regulation tags that can be widely adopted. We propose proteolysis targeting chimeric small molecules (PROTACs) based on TMP that target E. coli dihydrofolate reductase (eDHFR) tagged fusion proteins with high affinity. Our proof-of-concept molecules covalently link TMP and pomalidomide (POM), a ligand for the E3 ligase Cereblon. A lead compound, TMP- POM 7c robustly regulates diverse proteins, from optical reporter proteins, such as YFP and luciferase, to transcription factors and therapeutic membrane-bound proteins, such as CARs, in primary human T-cells. Optimization, characterization, and application of these compounds is needed, especially in terms of understanding the impact of linker length and composition, as well as pharmacokinetic properties, to lay the groundwork for a distributable prototype(s) that can be applied broadly in biomedical science. This approach represents a technological leap forward by uniting small molecule protein regulation of a versatile protein tag with fluorescence imaging probes and PET radiotracers for in vivo imaging.