Cisplatin, initially known as “Peyrone salt” was first synthesized by Michel Peyrone in 1845 [139]. In 1913, Alfred Werner proposed the square-planar configuration for the compound, and distinguished between cis and trans isomers, cisplatin and transplatin, respectively [140]. For this finding Alfred Werner won the Nobel Prize in Chemistry [140]. However, not until the 1960s was the anticancer property of cisplatin discovered. In 1965, Barnett Rosenberg carried out an experiment designed to monitor the effects of electromagnetic fields on bacterial growth [141]. The observation that bacteria grew filamentous, but had not divided, hinted that a compound produced during the experiment
could be a potential tumor-cell-growth inhibitor. The agent responsible for this effect was eventually identified as cis-diamminedichloridoplatinum(II) [142]. After successful demonstration of its anticancer activity, the FDA approved cisplatin in 1979 [143]. Since then, cisplatin and related platinum compounds (Figure 1.24) have been used to treat many carcinomas, including testicular, ovarian, bladder, colorectal, head and neck cancers, and lung cancers [144].
The cytotoxicity of cisplatin, the platinum compound used in this thesis work, is believed to originate from coordination to the nucleobases in DNA [145]. A general pathway for binding is shown in Figure 1.25 [146]. Once given as an intravenous drug, the labile chlorido ligands in cisplatin stay intact due to the high chloride concentration in blood plasma. Experimental results suggest that cisplatin is up taken by cells by both active transport and passive diffusion [147]. Once inside a cell, the chlorido ligands on cisplatin are displaced and the aquated, positively charged, activated form of cisplatin is generated. This active species then reacts with nucleic acids, and the resulting platinated adducts then lead to cell death [148].
Figure 1.25. A general pathway of cisplatin mechanism of action is shown [146]. Cisplatin forms different adducts with DNA (Figure 1.26A). It coordinates to the N7 position of purine bases, which is exposed in the major groove of DNA, to generate stable adducts (Figure 1.26B) [149]. In early studies, platination and characterization of cisplatin-treated salmon sperm DNA revealed 1,2-intrastrand cross-links as the major adducts, followed by 1,3-intrastrand and interstrand cross-links as the minor adducts [150]. The major 1,2-intrastrand cross-links are
cis-[Pt(NH3)2{d(GpG)}] (or cis-GpG; 47-50%) and cis-[Pt(NH3)2{d(ApG)}] (or cis-
ApG; 23-28%) [150]. The 1,3-intrastrand and interstrand cross-links are 8-10% percent of all adducts and monofunctional guanine comprises 2-3% of the damaged sites.
Cisplatin binding to DNA leads to significant structural effects. Crystallographic studies have shown that cisplatin binding bends the duplex
Figure 1.26. Cisplatin binding to nucleic acids is shown. A) Some of the cisplatin binding modes to a double helix are illustrated. B) The coordination of cisplatin to the N7 position of nucleobases is shown [151,152].
toward the major groove by about 35° [152]. The change in supercoiling of circular DNA suggests unwinding of the double helix [153]. NMR studies on a 1,3-intrastrand cross-linked adduct on DNA further supports the idea that the platination site is locally unwound and kinked [154]. Studies revealed that cisplatin adduct formation results in duplex destabilization and loss of helix stability [155,156]. Crystallographic studies revealed that the ammine ligands on cisplatin hydrogen bond to the oxygen atoms on the 5'-phosphate group, potentially stabilizing the adduct [152]. Collectively, these studies show that stable cisplatin adduct formation can induce structural changes to nucleic acids.
1.6.2. Kinetic Studies of Cisplatin-Nucleic Acid Interactions
Cisplatin can react with many targets such as nucleic acids, proteins, and small S-containing molecules in a cell [151]. Formation of different cisplatin adducts would depend on the reaction kinetics. The ligand-exchange reactions of square-planer compounds are slow [157]. This leads to kinetically controlled platination reactions of cisplatin binding to nucleic acids [158,159]. The proposed mechanism of cisplatin binding to double-stranded DNA is shown in Figure 1.27. The rate-limiting step for initial binding is hydrolysis of the first chlorido ligand. [160,161]. The aquated species then rapidly coordinates to DNA specifically at the N7 of purine bases. Loss of the second chlorido is the rate-determining step in the closure of the monoadduct [158].
Kinetic studies have given detailed insight to the primary reactive species, sequence-dependent reactivities, and the mechanism of cisplatin binding to nucleic acids [162]. Determination of reaction rates of phosphorothioate- substituted oligomers using 31P NMR showed that increased local concentrations of Pt(II) complexes, combined with higher mobility along the polymer backbone increases the platination rate [163,164]. Kinetic studies also demonstrated that target sites located at the ends of an oligomer platinate more slowly compared to the ones in the middle, indicating the importance of target location in platination reactions [165,166]. Flanking bases at the target site and nucleotide sequence also impact the platination rate [167,168]. Some of these observations were explained by a combined effect from electrostatic potential and N7 accessibility
Figure 1.27. Proposed mechanism of cisplatin binding to double helical DNA is shown [158].
of the target [169]. Kinetic studies have also shown that cisplatin discriminates between the 5' and 3' dGs in a dGG sequence for platination [170]. While the first platination is faster on 5' dG than on 3' dG, the chelation rates are opposite for the two Gs. The cisplatin preference for dAG adduct formation over dGA has also been explained by kinetic studies [171]. The higher rates of monoadduct formation, hydrolysis of the second chlorido ligand, and closure to form chelates on dAG compared to those of dGA have contributed to the increased platination of the former sequence. The platination kinetics also revealed that adduct
formation is dependent on whether the target sequence (dGG) is in a single- or double-stranded region [172].
Solution ionic strength and the type of cations present also determine the platination rates. Increasing pH, concentrations of salt, or divalent ions compared to monovalent ions reduce the platination rates [166,173,174]. Evaluation of salt- dependent kinetic data revealed that metal-complex binding releases cations from the nucleic-acid surface [173,175]. Interestingly, comparison of DNA and RNA reactions showed a more pronounced salt dependence in the platination rate of the latter [176]. Kinetic studies using full-length tRNA also showed a preference for G-C–rich and wobble base-pair regions in the platination reactions [177].
Platination kinetic studies can reveal much insight into nucleic acid-platinum complexes reactions. In addition, platination kinetics can be used to investigate the impact of RNA microenvironments on ligand interactions, which was a major focus of this thesis work. RNAs with natural nucleotide modifications (pseudouridine, Ψ, in H69) or altered sequences (the 790 loop) were selected to examine the impact of local environments on platination reactions. The evaluation of salt-dependent platination rates by the Brønsted-Debye-Hückel and polyelectrolyte theories allowed determination of the impact of global electrostatics on RNA-ligand interactions. The role of bulk solutions containing different monovalent and divalent cation concentrations or varying and pH values on selected RNA platination rates was determined. Platination kinetics were also applied to evaluate aminoglycoside-RNA interactions.
1.7 Specific Aims of the Research and Thesis Overview
Many studies have examined the characteristics of RNA molecular interactions. RNAs are central for various cellular functions; therefore, their interactions with small drug molecules can have a significant impact on cell biology. Characterization of RNA-drug interactions and investigation of their dynamics in response to changes in local environments are important. These studies are useful to identify unique RNA targets and to develop effective small molecule-based therapeutics.
This dissertation work was designed to investigate the kinetics of ribosomal RNA interactions with a platinum(II) complex, cisplatin. Many structural, biological, computational, and kinetic studies on DNA-cisplatin interactions have been reported to date [148,151,162,178]. Specifically, kinetic studies have provided valuable insight on the mechanism of drug binding [158,163- 168,173,175,176,179,180]. Many RNAs are also targeted by cisplatin [181-187]; however, kinetic studies on RNA-cisplatin interactions are largely unexplored and the binding mechanisms with RNA are largely unknown. Furthermore, salts, divalent ions, and pH play significant roles in biological interactions in a cell. Therefore, one of the main objectives of this thesis work was to carry out a broad kinetic study on cisplatin coordination to RNA. Analysis of the kinetic data with two different electrostatic models was done to reveal the impact of RNA microenvironments on drug binding. These findings are significant for expanding our knowledge on the mechanisms of platinum drug-RNA interactions. Importantly, this study reveals the impact of bulk conditions on RNA
electrostatics, which govern the platinum complexation reactions. Findings from the current study are valuable for identifying distinctive "druggable" RNA targets on bacterial ribosomes and their interactions with metal-based compounds. This knowledge could be further expanded to understand general cationic interactions with RNA.
The cationic cloud plays a significant role in RNA electrostatics, hence could impact structure, function, and ligand interactions [93,188-190]. The primary methods for studying these local environments are often based on computational approaches [85,191]. However, such theoretical methods may not reflect the true biological environments of molecules. Applications of some solution-based methods such as small-angle x-ray scattering and spectroscopic techniques to investigate the ionic cloud around DNA and RNA have been reported [192-195]. However, specific details on the RNA charge density and counterion displacement by ligand binding cannot be obtained from these studies. The use of chemical tools to interrogate RNA electrostatics is even rarer. Therefore, a gap in the field with respect to the development of chemical tools that can provide detailed information on microenvironments of structured RNA still exists. Another objective of this thesis work was to show the use of platination kinetics as an experimental tool to examine local environments of RNA, as it is sensitive to electrostatics illustrated by the binding mechanism. Platination kinetics was further applied as a tool to probe a known aminoglycoside-RNA interaction.
An overview of the dissertation work is as follows. Chapter 1 has introduced RNA and discussed its functions with respect to a key cellular activity, namely
protein synthesis. An introduction to ribosomes, RNA constructs studied in this thesis work, H69 and the 790 loop, RNA electrostatics, salt and pH influence on RNA, RNA-ligand interactions, and cisplatin was also provided in this chapter. The various techniques used in this thesis work and a description of the data analyses are provided in Chapter 2. Chapter 3 reports on cisplatin targeting of rRNA hairpins and site determination. The remaining chapters then focus on kinetic studies between cisplatin and the rRNA hairpins. The impact of monovalent cations on reaction rates (Chapter 4), the influence of monovalent and divalent cations on kinetics (Chapter 5), the sensitivity of cisplatin-ribosomal RNA binding rates to pH (Chapter 6), and probing aminoglycoside-ribosomal RNA interactions using cisplatin kinetics (Chapter 7) are reported. Finally, Chapter 8 gives some overall conclusions of the study and future directions.
CHAPTER 2
BIOCHEMICAL METHODS, KINETIC MEASURMENTS, AND DATA
ANAYSIS
A variety of biochemical methods and analytical/biophysical approaches are used in RNA research. This chapter provides a detailed background on the biochemical and biophysical techniques used to study cisplatin binding to ribosomal RNA. Data analysis methods used to determine platination rates and electrostatics of rRNA will be discussed in this chapter; whereas, specific experimental details will be given at the end of each section.
2.1 Matrix Assisted Laser Desorption Ionization Mass Spectrometry