Browsing by Subject "medicinal chemistry"

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    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.
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    Synthesis and Evaluation of a Metabolically Stabilized Analog and Conjugates of Pironetin
    (2019-08) Coulup, Sara
    Molecules that bind to tubulin and disrupt tubulin dynamics are known as microtubule targeting agents. Treatment with a microtubule targeting agent leads to cell cycle arrest followed by apoptosis. Tubulin inhibitors have been highly effective in the clinical treatment of a variety of tumors and are being investigated for treatment of several other diseases. Currently, all FDA approved microtubule inhibitors bind to β-tubulin. Given the overall success of tubulin-binding agents in anticancer chemotherapy, α-tubulin is an attractive and unexplored target. Herein, we will discuss the natural product pironetin, the only compound known to bind α-tubulin. Despite the potent in vitro activity against ovarian cancer cell lines both sensitive and resistant to current chemotherapeutics, pironetin was only marginally effective at high doses in mice and resulted in severe weight loss, indicating poor pharmacokinetic/pharmacodynamic (PK/PD) properties and significant off target toxicities. The research presented in this dissertation seeks to improve the therapeutic properties of pironetin by addressing the PK/PD and off target binding concerns. In Chapter 2, we determined that pironetin has a short half-life in liver microsomes and identified pironetin’s major site of metabolism in human liver microsomes to be the unconjugated olefin utilizing tandem mass spectrometry. We then confirmed the identity of the major metabolite as epoxypironetin through semi-synthesis and identified a very minor metabolite, demethylpironetin, with similar potency as pironetin. With the knowledge that the unconjugated olefin is the major site of metabolism, we sought to block this site through total synthesis. We thus completed the total synthesis of 4-fluorophenyldemethylpironetin (19 linear steps, 31 total steps) with the goal of improving the metabolic stability while maintaining potency (Chapter 3). This represents the first total synthesis of demethylpironetin analogs. Finally, in Chapter 4 we describe our efforts to establish a proof-of-concept method for the targeted delivery of pironetin to ovarian cancer cells by targeting the folate receptor. Our results suggest that pironetin conjugates do not enter the cell in a folate receptor mediated manner, therefore necessitating exploration of addition means of targeting for pironetin delivery.