Browsing by Subject "assay development"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    Biochemical and Biophysical Assay Development for Screening and Characterization of Small Molecules and Synthesized Analogues Targeting Human Cytosine Deaminases
    (2023-01) Grillo, Michael
    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.
  • Thumbnail Image
    Item
    Development of biophysical and Mass Spectrometry Assays for APOBEC3 cytosine deaminases: discovery and validation of cnvalent and non-covalent ligands
    (2022-11) Jones, Katherine
    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.