Optimization of and inducible control strategies for tag-targeted trimethoprim protacs

The National Cancer Institute awarded $110,228 to the University of Pennsylvania on February 12, 2027, under the Cancer Research Manpower program (CFDA 93.398) to support optimization and inducible control strategies for tag-targeted trimethoprim PROTACs.

The award funds research development of proteolysis targeting chimeras (PROTACs)—small molecule degraders that leverage proximity-induced pharmacology to target protein elimination. The University of Pennsylvania lab has developed E. coli dihydrofolate reductase (EDHFR)-targeting trimethoprim-based PROTACs to regulate EDHFR-tagged proteins, with integrated multimodal imaging capability enabling noninvasive in vitro and in vivo characterization of drug properties. The work addresses challenges in predicting and rationally optimizing PROTAC properties including potency, cell permeability, and pharmacokinetics. Research aims include establishing a novel iterative approach integrating machine learning to advance PROTAC development for targeted therapy across various diseases, particularly cancers.

Performance occurs at the University of Pennsylvania in Philadelphia, Pennsylvania, with work extending from February 12, 2027, through August 31, 2030. The award is classified as a Project Grant under the Cancer Research Manpower program, which supports biomedical training and research opportunities for individuals pursuing careers in basic, clinical, and prevention research relevant to the National Cancer Program.

Abstract

Conditional and reversible strategies to artificially regulate protein expression are invaluable to understanding and controlling protein function. Importantly, tunable regulation of an imaging-compatible protein tag could be transformative for a variety of in vitro and in vivo applications, including cellular medicines such as chimeric antigen receptor (car)-T therapy. Proteolysis targeting chimeras (PROTACS) are a well-known example of small molecule degraders that rely on proximity-induced pharmacology to hijack the cellular machinery responsible for protein recycling and eliminate proteins of interest. They have emerged as a promising approach for targeted therapy in various diseases, particularly cancers; current clinical molecular degraders that target the estrogen or androgen receptor are being investigated for the treatment of breast and prostate cancer, respectively. Our lab has developed E. Coli dihydrofolate reductase (EDHFR)-targeting trimethoprim (TMP)- based PROTACS to regulate EDHFR-tagged proteins. Uniquely, the EDHFR/TMP system can be used for both protein control and multimodal imaging, allowing for noninvasive in vitro and in vivo characterization of drug properties and thus furthering our understanding of degrader development. The extensive methods of characterization also support efforts to discover more disease-relevant proteins amenable to degradation. While PROTACS have several advantages over traditional inhibitors, such as the ability to exert therapeutic effects at sub-stoichiometric doses and degrade targets that lack enzymatic activity or allosteric regulatory sites, their large, untraditional structures have led to challenges in predicting and rationally optimizing properties such as potency, cell permeability, and pharmacokinetics. Aim 1 of this work will establish a novel, iterative approach integrating machine learning, automated solid-state compound synthesis and purification, and biological screening to explore broad chemical space for the linker region of these molecules and address current challenges hampering rational and accelerated development of PROTACS. While PROTACS have demonstrated robust protein degradation and success in clinical trials, a lack of external control over their function remains a limitation to their applications as chemical tools and therapeutics. Aim 2 will explore conditional regulation strategies for imparting spatiotemporal control to these molecules. This is particularly promising for achieving precise control of genetic medicines, such as car-T cells. Spatiotemporal control of proteins that are the basis of cancer-targeted cellular therapies could mitigate adverse effects and toxicity of these therapies. Ultimately, this project aims to provide the foundation for new approaches to the rational development of optimized bifunctional molecules, as well as conditional control strategies for their use. Imageable, tag-targeted molecules that can regulate protein homeostasis will provide tools to probe and understand disease-relevant proteins.