APOBEC3 (APOBEC3A-APOBEC3H) enzymes catalyze single-stranded (ss)DNA cytosine-to-uracil (C-to-U) deamination as a function of innate immune defense against foreign DNA. Host cellular protection results from APOBEC3-catalyzed lethal mutagenesis of the offending genome. When misregulated, however, APOBEC3 enzymes have been demonstrated to drive the genetic evolution of numerous cancers and HIV-1. Specifically, sub-lethal levels of APOBEC3D/F/G/H-catalyzed mutation can enable HIV-1 escape from immune defense and antiretroviral therapies. Moreover, APOBEC3B over-expression in breast, bladder, cervical, lung, and head/neck cancers generates high levels of C-to-U mutation, which drives tumor formation, metastasis, and chemotherapeutic resistance. Thus, small molecule inhibition of APOBEC3-catalyzed deamination may provide a novel strategy for HIV-1 and cancer drug development. This thesis highlights efforts to discover small molecule inhibitors of the APOBEC3s through high-throughput screening (HTS) and chemical synthesis. Chapter 2 discusses an HTS of 168,192 compounds against APOBEC3B and APOBEC3G. In this effort, MN23 was discovered as a potent inhibitor of APOBEC3B (IC50 = 150 nM) and APOBEC3G (IC50 = 5.5 µM). Chapter 2 also reports a novel synthesis of MN23, and preliminary efforts to elucidate its mechanism of inhibition. Chapter 3 presents a class of covalent APOBEC3G-specific inhibitors based on a 1,2,4-triazole-3-thiol substructure. This compound class is predicted to inhibit APOBEC3G by covalently binding C321, forcing an inhibitory conformational change within the enzyme active site. Chapter 4 reports the discovery of a novel APOBEC3G inhibitory chemotype, which was discovered from the deconvolution of an impure HTS hit. In this effort, we also identified a previously unreported Pan Assay Interference Scaffold (PAINS), and characterized the mechanism by which compounds of this class undergo oxidative decomposition. Finally, Chapter 5 describes how benzthiazolinone-based APOBEC3 inhibitors are being developed into probes of APOBEC3 structure and function. Brief descriptions of two unrelated projects performed concurrent with these studies are also detailed in the Appendices.
University of Minnesota Ph.D. dissertation. September 2015. Major: Medicinal Chemistry. Advisor: Daniel Harki. 1 computer file (PDF); xiv, 251 pages.
High-throughput Screening and Chemical Synthesis for the Discovery of APOBEC3 DNA Cytosine Deaminase Inhibitors.
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