Date of Award
Christine S. Chow
With the discovery of cisplatin in the 1960s, it has been widely studied as a precursor for anticancer drug development. Despite its effectiveness against certain cancers, clinical usage of cisplatin is restricted by a number of side effects and resistance. In the past decade, scientists have been exploring biologically important ligands such as sugar derivatives in the hope of overcoming such challenges. Attachment of a sugar moiety could facilitate lower accumulation of platinum drugs in the body as well as enhance cellular uptake. In this study, a carbohydrate-linked cisplatin analog, cis-dichlorido[(2-β-D-glucopyranosidyl)propane-1,3-diammine]platinum (5) has been studied. The aim was to evaluate the impact of the carbohydrate moiety on the reaction kinetics with nucleosides, target site identification, structural impacts on oligonucleotides, and selective cancer cell targeting. All of the experiments in this thesis work were carried out in comparison to the parent compound cisplatin. Compound 5 was synthesized and a reactivity study was carried out with deoxy-/ribonucleosides and oligonucleotides using high performance liquid chromatography (HPLC) and liquid chromatography mass spectrometry (LC-MS). A series of gel-based kinetic experiment was carried out comparing 5 and cisplatin with DNA and RNA oligos, with mono- and bis-activated complexes. A salt dependence kinetic study was also carried out to understand the contribution of electrostatics on the reactivity. A study was done to analyze the effects of compound 5 on DNA duplex structure by measuring the bending angles. Furthermore, probing studies on ribosomal RNA (rRNA) showed compound 5 localization on rRNA. The potency of the compound was assessed by MTT assays. Overall, mono-activated compound 5 showed preferred binding to G over dG, which is opposite of the preference of cisplatin for dG. A similar trend was observed for mono-activated 5 at the oligonucleotide level, in which RNA binding was preferred over DNA. However, in all the experiments, the reaction rate for mono- or bis-activated 5 was lower (≥2.5-fold) than that of aquated cisplatin. Data from the kinetic experiments suggest that the bulky carbohydrate ligand may play a role in reducing the reactivity of compound 5. Salt dependence kinetic data suggested the RNA binding preference of 5 may come from its ability to form additional hydrogen-bonding interactions with the target compared to cisplatin. Therefore, glycoconjugation alters target specificity of the platinum compound. Furthermore, compound 5 formed platinum adducts that bend the DNA structure in a manner similar to cisplatin. Cell-based assays showed that 5 has selectivity towards glucose transporter (GLUT) overexpressing cancer cells (DU145) over normal cells (RWPE1). Overall, these findings support the potential of using glycoconjugated platinum complexes as lead compounds for anticancer drug development with controlled activity and target selectivity.
Electrostatics play an important role in RNA-ligand interactions. Positively charged drug molecules and other ligands are attracted to the negatively charged RNA. Monovalent and divalent ions affect the electrostatic properties and influence these interactions; however, few methods exist to study these interactions in solution. This work focused on the use of cationic transition metal complexes as tools to study the electrostatic contributions of drug-RNA interactions. More specifically, positively charged mono-activated cisplatin and small model rRNA constructs were employed. The rates of the platination were determined by using gel shift analysis in the presence of different aminoglycosides. The rates of platination with RNA were shown to decrease in the presence of aminoglycosides, suggesting a competition between cisplatin and aminoglycosides to bind to the RNA. This work demonstrated the utility of monoaquated cisplatin kinetics as a tool to study RNA-aminoglycoside interactions. Overall, these findings are important for providing details on drug-RNA interactions, improving cationic drugs for nucleic acids, identifying unique RNA drug targets, and developing tools to investigate RNA microenvironments.
Thalalla Gamage, Supuni Duneeshya Kamal, "Insights Into Nucleic Acid-Platinum(ii) Compound Interactions And Structural Impacts" (2019). Wayne State University Dissertations. 2189.