Biochemical and Biophysical Assay Development for Screening and Characterization of Small Molecules and Synthesized Analogues Targeting Human Cytosine Deaminases

Abstract

The APOBEC3 family of enzymes converts cytosine to uracil in single-stranded DNA as a part of the innate immune defense against viruses. A large body of work has demonstrated that when dysregulated, APOBEC3s contribute to mutagenesis of somatic DNA in cancer. These mutational events lead to poor clinical outcomes such as tumor recurrence, metastasis, and therapeutic resistance. Because of this, we are interested in targeting APOBEC3s for inhibition by small molecules with the goal of improving the outcome of treatment with current cancer therapies. Chapter 1 introduces APOBEC3s as targets of interest with commentary on current and potential biochemical, biophysical, cellular, and in vivo assay technologies to evaluate potential inhibitors. Chapter 2 discusses careful in silico reconstruction of APOBEC3B followed by molecular dynamics simulations and druggability analysis identifying putative allosteric sites. Virtual screening was performed, and compounds were validated in biochemical and biophysical assays to serve as potential starting points for hit to lead optimization. Chapter 3 focuses on the development of sensitive biophysical assays and implementation in fragment screening. One fragment was validated during triage and served as a starting point for preliminary structure-activity relationship studies on two potentially divergent chemical series. Chapter 4 discusses the development of a real-time fluorescence-based activity assay for human cytidine deaminase utilizing a fluorogenic substrate. This assay was validated with known small molecule inhibitors and implemented in a fragment screen to discover novel non-nucleoside inhibitors of cytidine deaminase. The long-term goal of this work is to apply the same technology to measure APOBEC3 activity. Chapter 5 outlines several attempts at synthesizing a rationally designed covalent sulfur (VI) fluoride exchange probe targeting conserved hydroxyl-containing residues in the APOBEC3 active site. A variety of standard, as well as novel, approaches to nucleoside chemistry were explored with the goal of eventually incorporating a covalent warhead into a DNA oligonucleotide. Finally, Appendix A describes attempts at expressing and purifying A3B containing 5-fluorotryptophan as a tool for protein-observed 19F-NMR experiments.