Identification, Development, and Evaluation of Small-Molecule Modulators of Nucleotide Metabolism

This dissertation describes the identification, development, and evaluation of small- molecule modulators of nucleotide metabolism. Nucleotides, well-known for their role as the “backbone” of DNA, are essential for a variety of cell processes including energy transfer, formation of lipid membranes, and DNA and RNA synthesis. Small molecule-mediated inhibition of enzymes involved in nucleotide metabolism has shown promise in various therapeutic settings, and our group has demonstrated that inhibition of deoxycytidine kinase combined with perturbations to other nucleotide-producing enzymes can effectively treat acute lymphoblastic leukemia in mouse models.Chapter One details the development and evaluation of inhibitors of deoxycytidine kinase. Expanding upon a scaffold previously identified by our group, structure-activity-relationship studies were undertaken in pursuit of a clinically-viable inhibitor. With the goal of eliminating the presence of a stereocenter within our lead compound while maintaining low- nanomolar affinity for deoxycytidine kinase, a series of analogs containing a gem-dimethyl moiety was produced. In addition, the preclinical pharmacology of the lead compound, DI-87, was evaluated with the aid of PET imaging.Chapter Two describes the development and application of a cell-based metabolic modifier screening platform that leverages the redundancy in pyrimidine metabolism for the discovery of selective uridine monophosphate biosynthesis modulators. In evaluating a library of protein kinase inhibitors, multiple compounds which possess nucleotide metabolism modifying activity were identified. The JNK inhibitor JNK-IN-8 was found to potently inhibit nucleoside transport and engage ENT1. Additionally, the PDK1 inhibitor OSU-03012 and the RAF inhibitor TAK-632 were shown to inhibit the therapeutically relevant enzyme DHODH, and their affinities were unambiguously confirmed through in vitro assays and co-crystallization with human DHODH