High-throughput Screening and Chemical Synthesis for the Discovery of APOBEC3 DNA Cytosine Deaminase Inhibitors

University of Minnesota Ph.D. dissertation. September 2015. Major: Medicinal Chemistry. Advisor: Daniel Harki. 1 computer file (PDF); xiv, 251 pages.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