A Bioorthogonal Approach to Studying Platinum Drug Targets Using Modified Platinum (II) Complexes With Alkyne- and Azide-Containing Handles

The compound cisplatin and related FDA-approved Pt(II) therapeutics have been used ubiquitously to treat a variety of cancers since the late 1970’s. Despite the success of Pt therapeutics, their use is limited by undesirable side-effects such as hearing loss, peripheral neuropathy, and severe kidney damage, in addition to intrinsic and acquired resistances. Despite the need to further study and understand this important class of compounds, we lack a comprehensive understanding of global Pt drug targets which would lend vital insight into the molecular mechanisms of action of Pt. This dissertation describes a novel technology using modified Pt(II) drug analogues which contain bioorthogonally reactive handles to study Pt drug targets using post-binding covalent modifications. Chapter I describes the history of Pt anticancer therapeutics and early efforts made to elucidate their mechanism of activity. New methods to track and analyze Pt in biological systems are needed to further study Pt therapeutics, for which we have chosen to modify Pt compounds to contain small, minimally invasive bioorthogonally reactive handles. These handles allow for subsequent fluorescent detection or isolation via the azide-alkyne cycloaddition reaction, or prototypical “click” reaction. Chapter II describes the synthesis of the first difunctional click-modified Pt(II) complex capable of post-binding covalent click modification. Both rRNA and tRNA were identified as targets of Pt in vivo. Chapter III describes the use of another azide-appended Pt chelate complex in post-binding target studies. Click labeling of Pt-bound model protein was demonstrated, along with the further characterization of Pt-rRNA targets, which are shown to be relatively long-lived in Saccharomyces cerevisiae. Chapter IV describes the first alkyne-appended Pt(II) complex capable of post-binding click modification, which undergoes a re-arrangement to form the stable N-sulfonyl amide upon fluorescent “turn-on” ligation with dansyl azide. Chapter V reports the azide analogue of the aforementioned Pt-alkyne complex and demonstrates fluorescent localization studies in triple-negative human breast cancer cell lines, observing significant nucleolar and nuclear Pt localization. Finally, Chapter VI summarizes miscellaneous work in developing additional syntheses to generate small, azide-appended ethylenediamine and peptide-coupled alkyne derivatives of cisplatin. This dissertation includes previously published and unpublished co-authored material.10000-01-0