Date of Award
Doctor of Philosophy (PhD)
Mark A. Sellmyer
Detecting and manipulating cells and their interactions is a broad goal of chemical biology with potential translational impact. For example, detecting live bacteria in vertebrate animals is a crucial advance for infection diagnosis. Similarly, monitoring engineered mammalian cells is important when developing new cellular therapies. Beyond detecting a specific cell population, controlling cell-cell interactions is a fast- developing research area with practical applications. Immune-mediated therapeutics such as checkpoint inhibitors drive the interaction of immune cells with cancer cells. Similarly, chimeric antigen receptor (CAR) T cells use combinations of protein domains to build a sense-and-respond architecture into a therapeutic paradigm based on cell-cell interactions. Still, challenges remain for maximizing the potential of immunotherapies including on-target, off-tumor toxicity. Development of molecular technologies to fine- tune temporal and spatial dynamics of protein expression could bolster efficacy while reducing toxicity. Here, small-molecule systems may offer unique value in both detecting and manipulating cell interactions as they are facile to iterate, easily permeate cells, and may be less expensive than other modalities. Trimethoprim (TMP) is an antibiotic that reversibly inhibits bacterial dihydrofolate reductase (DHFR) with 10,000-fold selectivity over the mammalian DHFR homologue. Such a large therapeutic window renders TMP inert in mammalian cells and makes it an ideal molecular chassis for building new chemical biology platforms. In this thesis, I detail advances in two TMP- based molecular tools. First, I describe a two-cell system that can regulate protein expression based on the selective activation of a TMP prodrug. Initial proof-of-concept studies employ a bacterial nitroreductase enzyme while later studies use a human cancer target, fibroblast activation protein, for the catalytic uncaging of a steric protecting group on the TMP. Second, I describe the development and application of a TMP-based PET radiotracer, [11C]-TMP, and determine whether it can image drug- resistant bacteria. In vitro and bioinformatic approaches uncover a DHFR redundancy pattern that allows TMP-resistant bacteria to be imaged by [11C]-TMP. Together, these tools may serve as powerful approaches to both image pathologic processes and manipulate future engineered molecular technologies.
Jacome, Daniel Alejandro, "Imaging And Manipulating Cells And Their Interactions With Trimethoprim-Based Tools" (2022). Publicly Accessible Penn Dissertations. 5619.