Cellular RNA targeting by platinum (II) anticancer therapeutics.

摘要

Cis-diamminedichloroplatinum (II), or cisplatin, is a widely prescribed anticancer compound, currently one of only three platinum (II) complexes FDA approved for cancer treatment. Despite its widespread use, we lack a comprehensive picture of global drug targets, which would lend valuable insights into the molecular mechanisms of action and resistance in different tissues. Drug binding to genomic DNA is an accepted cause of downstream apoptotic signaling, but less than 10% of Pt (in the case of cisplatin) accumulates within genomic DNA. Non-genomic contributions to cisplatin's therapeutic action are also under active investigation. In particular, cisplatin treatment can disrupt RNA-based processes such as splicing and translation. Pt(II) targeting of non-DNA species such as RNA may contribute to or sensitize a cell to the downstream effects of this drug, including the induction of apoptosis. Chapter I summarizes the activity profile of Pt(II) therapeutics, describing cellular uptake, cellular localization, incidences of Pt(II) accumulation within RNA, and RNA processes affected following drug treatment. Chapter II reports our thorough investigation of the distribution of Pt species throughout messenger and ribosomal RNA, with the discovery that Saccharomyces cerevisiae ribosomes act as a de facto cellular Pt sponge. In Chapter III, we report the synthesis of an azide-functionalized platinum (II) species, picazoplatin, for post-treatment click labeling and isolation of drug targets in vivo. Picazoplatin was designed to circumvent mislocalization and misprocessing of Pt typically encountered when trying to track small molecules tethered to large, charged fluorophores. This chapter contains several proof-of-principle studies validating the use of this class of reagents for future purification and sequencing of Pt-bound nucleic acids. Chapter IV describes the first application of the click-capable Pt reagent technology: the demonstration of significant in-gel fluorescent detection of Pt-bound ribosomal RNA and transfer RNA extracted from picazoplatin-treated S. cerevisiae and the first evidence that cellular tRNA is a platinum substrate. Chapter V summarizes these data, which suggest a potential ribotoxic mechanism for cisplatin cytotoxicity and broadly describe a convenient click chemistry methodology that can be applied to identify other metal or covalent modification-based drug targets. This dissertation includes previously published and unpublished co-authored material.