Development of biophysical and Mass Spectrometry Assays for APOBEC3 cytosine deaminases: discovery and validation of cnvalent and non-covalent ligands

Abstract

APOBEC3 enzymes are cytosine deaminase enzymes that catalyze the conversion of a cytosine base to uracil in single-stranded DNA. There are seven members of the APOBEC3 family – A3A/B/C/DE/F/G/H. APOBEC3 proteins are a part of the innate immune defense system against viral infection, and mutate the viral genome to restrict replication. Multiple studies have demonstrated that APOBEC3 deaminase activity contributes to mutations in the somatic genome, thereby increasing the mutational burden in tumor genomes and leading to genomic instability, tumor heterogenity, and the development of therapeutic resistance. APOBEC3 proteins are therefore important drug targets for the development of inhibitors that can be used in combination with traditional cancer therapies to increase their efficacy. This thesis will discuss various approaches to discover and develop APOBEC3 ligands. Chapter 2 will discuss an effort at utilizing in silico screening followed by in vitro validation for the discovery of novel A3B inhibitors. Molecular dynamics simulations identified a novel putative allosteric site on A3B; next, the virtual screening following by biochemical assay screening resulted in the discovery of novel A3B ligands. Chapter 3 discusses a novel approach to A3A ligand discovery through fragment-based disulfide tethering, which yielded several fragments that could prevent DNA binding or inhibit wild-type A3A deaminase activity. Chapter 4 presents a novel assay developed for the detection of non-specific thiol binding of covalent compounds. The assay, termed ALARM MSPS (A La Assay to detect Reactive Molecules via Mass Spectrometry Peptide Sequencing), can be utilized to measure any covalent compound and its non-specific protein thiol reactivity through isotopic labeling. Appendix A describes a previously undescribed phenomenon where the A3A inactivating mutation E72Q destabilizes protein tertiary structure, where the E72A inactivating mutation does not. Appendix B discusses efforts at resynthesizing and retesting hit compounds from a cellular base editing screen that was performed to identify inhibitors of A3B.