Characterizing the nucleosome site selectivity, adduct structures and impact on chromatin for a variety of metal-based anticancer compounds and structural studies of nucleosome-linker histone interactions

Abstract

The enormous length of DNA molecules is managed in the nucleus of eukaryotic cells by wrapping the double helix through histone protein association to form chromatin. The basic repeating unit of chromatin is the nucleosome. Due to this packaging, nucleosomal DNA and histones have different conformations and accessibility compared to that of naked DNA or chaperone-bound histones. The structure and dynamics of chromatin are important for many cellular functions. Different nuclear factors bind to nucleosomes to regulate chromatin dynamics and signal downstream cellular processes, like DNA replication and transcription. As a consequence, nucleosomes and their binding partners can be specifically targeted to modulate these functions. This is especially relevant to cancer cell function, whereby numerous epigenetic differences, such as alterations in nucleosome positioning, between transformed and healthy cells result from extensive changes in gene expression and the frequency of DNA replication (cell division). Distinguishing epigenetic features of cancer cells could be potentially exploited through the design of site-specific chromatin-targeting anticancer agents. Because of the severe side effects and resistance problems associated with platinum anticancer drugs, many different compounds based on alternative heavy metals have been explored as potential therapeutic agents and many more are presently under investigation. However, the cellular targets and mechanisms of action for these newer agents are largely unknown. Based on studies with cisplatin and other platinum drugs, DNA is generally assumed to be the cellular target for reactive metal-based compounds. However, this thesis work and other recent evidence show that proteins are the main targets for at least some of these compounds. Since the nucleosome contains both DNA and protein components and is an important therapeutic target, it is useful for investigating the DNA- versus protein-binding preferences of different metal-based agents. We investigated the site selectivity, adduct structures and impact on chromatin for many different metal-based compounds, including ruthenium, osmium, gold and rhodium agents. This involved a broad spectrum of biochemical, X-ray crystallographic and electron microscopic (EM) techniques in conjunction with collaborations on cellular, analytical chemistry and computational approaches. The striking discovery is that, with the exception of one class of ruthenium agent, the compounds investigated show a preference for binding to histone protein sites as opposed to the DNA of the nucleosome. Moreover, by comparing two structurally similar ruthenium compounds, one a cytotoxic anti-primary tumor agent and the other an effectively non-cytotoxic antimetastasis agent, we could show the basis for their differential DNA versus histone protein targeting and consequently linkages to their distinct cellular impact. For the compounds that preferentially form adducts at histone protein sites, we also discovered that there are two distinct…