The uracil base excision repair (BER) pathway plays a pivotal role within the cell by removing aberrantly incorporated uracils from the genome. While it would seem counterintuitive to inhibit a pathway that works to maintain the fidelity of the genome, uracil BER has recently arisen as a potential therapeutic target in a variety of human diseases. In addition, uracil BER is important in the cell killing mechanism of 5-fluorouracil (5-FU), suggesting that inhibitors of this pathway would enhance the efficacy of this widely used anticancer drug. These potential targets for biomedical intervention have become apparent from a basic understanding of the delicate balance of uracil nucleotide metabolism maintained within the cell and the consequences of disrupting this balance.

The first aim of this thesis is to identify enzymes of uracil BER that are important in human disease and 5-FU anticancer therapy. The second is to discover small molecule inhibitors that are effective against these enzymes. Human uracil DNA glycosylase (UNG) plays a pivotal role in both HIV transmission and antibody diversification, indicating that inhibitors of this enzyme would be beneficial for anti-HIV therapy and the treatment of severe allergies. A pyrollidine-based inhibitor of UNG was developed based on the structure of the highly dissociative glycosyl cation transition state of the UNG reaction, and a crystal structure of UNG complexed with this inhibitor and uracil was refined to 1.9 Å resolution. Using this structure, it was possible to design small molecule inhibitors of LNG with a bipartite pyrollidineluracil-anion motif.

In a second line of study, the budding yeast Saccharomyces cerevisiae was used to investigate the role of uracil BER enzymes in 5-FU toxicity. The 5-FU sensitivity of yeast strains that were genetic knockouts in these enzymes was determined and compared with that of the wild-type strain. This profiling revealed that apurinic/apyrimidinic endonuclease (Ape1) increased the sensitivity to 5-FU by 10-fold. Given this result, high-throughput screening was used to discover a novel class of high affinity small molecule inhibitors of human Ape1. These inhibitors provide useful templates for the design of new derivatives that target AP endonuclease.