Evaluation and Development of Metal-Binding Agents that Alter Copper Bioavailability

Abstract

Transition metal ions are required nutrients for many organisms but also potent toxins if misappropriated. Iron (Fe) plays a central role in the transport of oxygen, and other transition metals, such as copper (Cu) or zinc (Zn), are found in enzymes. It is critical for the human body to maintain careful control on both the levels of metals and their distribution to maintain healthy function. An imbalance in these metals such as an overload often leads to organ failure, while deficiencies result in other medical conditions like anemia, neutropenia, leukopenia. Metal imbalances have been implicated in neurodegenerative diseases, cancer and infections. This dissertation explores several strategies envisioned to alter the bioavailability of metal ions by using synthetic metal-binding agents targeted specifically for diseases where misappropriated metal ions are suspected of exacerbating cellular damage. In Chapter 1, we discuss chemical properties that influence the pharmacological outcome of a subset of metal-binding agents known as ionophores, and review several examples that have shown multiple pharmacological activities in metal-related diseases, with a particular focus on Cu. Chapter 2 describes results of a growth assay in which we screened small molecule Fe and Cu chelators to determine if altering the bioavailability of these essential metal ions inhibits growth of the fungal pathogen, Cryptococcus neoformans. Results show that select chelating agents that facilitate the increase of intracellular Cu levels inhibited growth of C. neoformans, while traditional metal sequestering agents had no effect on growth at the concentrations tested. In Chapter 3, various chemical properties of the select ligands that demonstrated Cu-dependent antifungal activity (8-hydroxyquinoline (8HQ), thiomaltol and pyrithione) were analyzed and compared to those of counterpart ligands that did not inhibit C. neoformans growth, namely clioquinol, maltol, pyridinol-n-oxide, deferiprone, and thiodeferiprone. The UV-vis spectroscopy of the Cu(II) and Cu(I) complexes of each ligand, along with calculation of their apparent binding affinities by competitive ligand titrations, are described. In addition, we determined octanol-water partition coefficients for the Cu(II) complexes and compare them with reported partition coefficients for the free ligands. An initial assessment of the reduction potential of Cu complexes of the select agents is also investigated in this chapter, along with an analysis of structural details from available crystallographic data.After identifying chelating agents that inhibit C. neoformans in a Cu-dependent manner, we take a closer look in Chapter 4 at 8HQ as a model Cu ionophore. In spite of its promising biological activity of, the metal-dependent toxicity of 8HQ extends to mammalian cells as well. To overcome this challenge, Chapter 4 describes the application of a prochelator strategy to manipulate host Cu in innate immune cells to fight microbial infection. QBP is a nontoxic protected form of 8HQ in which a pinanediol boronic ester blocks metal ion coordination by 8HQ. The prochelator, QBP, is deprotected via reactive oxygen species produced by activated macrophages, creating 8HQ and eliciting Cu-dependent killing of C. neoformans in vitro. Finally, Chapter 5 outlines the synthesis of multifunctional metal chelators that contain a masking group on a metal binding moiety that has been incorporated onto the structural framework of Aβ aggregate-imaging agents. Masking the metal binding site should prevent non-specific metal binding of the prochelator. Once activated to its unmasked form under conditions that mimic early Alzheimer's disease, the released chelator should complex metal ions. The design and rates of oxidation in response to hydrogen peroxide exposure along with their ability to interact with Cu are described in Chapter 5.