Investigating the binding mode of azide labelled derivatives of Hoechst 33258 by NMR, UV-Vis and IR spectroscopy

This thesis describes attempts to develop a method for studying the dynamics of dsDNA: small molecule ligand association and dissociation through 2D-IR spectroscopy. The strategy employed was to synthesise azide-bearing derivatives of the archetypal minor groove binding ligand, Hoechst 33258, with the azide acting as a reporter functional group. Chapter 1 describes both the state of the art, and also what is unknown regarding the processes of association and dissociation of small molecules from dsDNA, and how this project aims to gain a fuller understanding of the dynamics of association between dsDNA and a small molecule MGB. The clinical significance of minor groove binders is also discussed. Chapter 2 describes the synthesis of two azide-bearing derivatives of H33258 through an amide coupling strategy, and the significant problems encountered with the purification of these compounds. Future alternative pathways to these compounds are proposed. An investigation into the spectroscopic properties of the azide functional groups in the free compounds is also presented. Chapter 3 describes the investigation of the utility of the azide functional group through a comparative study of the thermal dissociation of these compounds from dsDNA. It was found that the utility of the azide was dependent on the position of the azide within the molecule, and is sensitive to the changes in solvation of the minor groove of dsDNA. One compound in particular exhibited a marked change in both the shape and the intensity of the azide absorption band and was commensurate with the melting temperature, Tm of the complex. Chapter 4 describes the investigation into the structural origins of the marked difference in the azide absorption band of one MGB when in complex with a dsDNA oligomer through structural characterization of this complex by NMR spectroscopy. It was found that the azide functional group is in close proximity to the exocyclic amine of guanosine, and this specific interaction is proposed to give rise to the observed changes in the azide absorption band. The orientation of the molecule was found to be opposite to that reported for H33258, a reason why this is the case is also proposed.This thesis describes attempts to develop a method for studying the dynamics of dsDNA: small molecule ligand association and dissociation through 2D-IR spectroscopy. The strategy employed was to synthesise azide-bearing derivatives of the archetypal minor groove binding ligand, Hoechst 33258, with the azide acting as a reporter functional group. Chapter 1 describes both the state of the art, and also what is unknown regarding the processes of association and dissociation of small molecules from dsDNA, and how this project aims to gain a fuller understanding of the dynamics of association between dsDNA and a small molecule MGB. The clinical significance of minor groove binders is also discussed. Chapter 2 describes the synthesis of two azide-bearing derivatives of H33258 through an amide coupling strategy, and the significant problems encountered with the purification of these compounds. Future alternative pathways to these compounds are proposed. An investigation into the spectroscopic properties of the azide functional groups in the free compounds is also presented. Chapter 3 describes the investigation of the utility of the azide functional group through a comparative study of the thermal dissociation of these compounds from dsDNA. It was found that the utility of the azide was dependent on the position of the azide within the molecule, and is sensitive to the changes in solvation of the minor groove of dsDNA. One compound in particular exhibited a marked change in both the shape and the intensity of the azide absorption band and was commensurate with the melting temperature, Tm of the complex. Chapter 4 describes the investigation into the structural origins of the marked difference in the azide absorption band of one MGB when in complex with a dsDNA oligomer through structural characterization of this complex by NMR spectroscopy. It was found that the azide functional group is in close proximity to the exocyclic amine of guanosine, and this specific interaction is proposed to give rise to the observed changes in the azide absorption band. The orientation of the molecule was found to be opposite to that reported for H33258, a reason why this is the case is also proposed

Targeting Duplex and Higher Order Nucleic Acids Using Neomycin / Neomycin-Hoechst 33258 Conjugates

Small molecules have provided means to combat many diseases caused by pathogens. In the last six decades, advances in the structural and biological understanding of nucleic acid functions have led to rational drug design programs in order to solve current therapeutic challenges. The work described in this dissertation addresses discovery of aminoglycosides as novel binders to G-quadruplex nucleic acids. Understanding of effect of linker length on the B-DNA binding of a series of Hoechst 33258 derivatives have been provided. Novel Hoechst 33258 based monobenzimidazoles have been synthesized and their biological properties have been compared with the bisbenzimidazoles. The bisbenzimidazoles have been designed to be useful towards click chemistry applications. These clickable Hoechst 33258 derivatives were then used to prepare neomycin-Hoechst 33258 conjugates with varied linker spacing between them. The binding studies of these novel neomycin-Hoechst 33258 conjugates show intercalative binding of bisbenzimidazole moiety of the ligand to an RNA duplex. Finally, the novel neomycin-Hoechst 33258 conjugates have been screened against G-quadruplex forming promoter sequences and biophysical characterization to its binding has been provided. Overall, these studies have led to synthesis of novel small molecules that bind to various nucleic acids and their new binding modes have been discovered. The studies presented here are expected to help in the design of novel therapeutics

Drug-targetting of duplex and quadruplex DNA

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies

Drug-targetting of duplex and quadruplex DNA

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies

Understanding the Recognition of Mixed Sequence DNA through Minor Groove Binding Compounds

The broad range of diseases controlled by transcription factors (TFs) and their potential abilities to modulate gene expression have led to an emerging interest in the development of small molecules that target TF-DNA complexes. Still, there is only a limited number of types of designed small molecules that show strong and sequence-specific binding to DNA along with good cellular uptake properties for therapeutic use. Most of the successful nuclear stains or therapeutic agents that bind non-covalently in the minor groove of DNA are AT specific. Synthesis of novel compounds to better target the mixed AT/GC base pair (bp) sequences with a broad range of applications like targeting TF is a daunting task. Our novel heterocyclic cation, DB2277, contains the aza-benzimidazole group (aza-BI) that specifically recognizes the single GC bp interspersed between AT bp sequences in the minor groove of DNA. NMR spectroscopy revealed the presence of major and minor binding species in the DB2277 complex with AAAGTTT type of DNA. NMR exchange dynamics have shown that the exchange between major and minor species is much faster than the compound’s dissociation from the complex, as determined using surface plasmon resonance (SPR). To understand the molecular basis of recognition of mixed bp sequences and to acquire ideas to design new sequence-specific compounds, structural information of the DB2277-DNA complex is essential. Experimental structure of the unique and selective binding orientation of DB2277 with “AAGATA” binding site of DNA has been obtained using high-resolution NMR and molecular dynamics (MD) simulations, which suggests the involvement of two specific and strong H-bonds in recognition of the central GC bp. Extended MD calculations have shown dynamic water-mediated H-bond contacts between amidine of DB2277 and the bases at the floor of the minor groove and 180° rotations of the phenyl linked to a flexible linker (OCH2) in a bound compound for the first time. Therefore, designing additional compounds with the ability to recognize a vast array of biologically important DNA sequences is essential for extending the use of new heterocyclic compounds in therapeutic applications in the future

Diagnosis and Inhibition Tools in Medicinal Chemistry

Cell surface saccharides are involved in a variety of essential biological events. Fluorescent sensors for saccharides can be used for detection, diagnosis, analysis and monitoring of pathological processes. The boronic acid functional group is known to bind strongly and reversibly to compounds with diol groups, which are commonly found on saccharides. Sensors that have been developed for the purpose of saccharide recognition have shown great potential. However, they are very hydrophobic and this lack of essential water-solubility makes them useful in biological applications. The first section of this dissertation details the process of developing water-soluble saccharide sensors that change fluorescent properties upon binding to saccharides. The second section of the dissertation focuses on the development of DNA-minor groove binders as antiparasitical agents. Parasitical diseases comprise some of the world’s largest health problems and yet current medication and treatments for these parasitical diseases are often difficult to administer, costly to the patients, and have disruptive side effects. Worse yet, these parasites are developing drug resistance, thus creating an urgent need for new treatments. Dicationic molecules constitute a class of antimicrobial drug candidates that possess high activity against various parasites. The second section details the development of a series of di-cationic agents that were then screened in in vitro activities against parasitical species

Copper(II) Phenanthrene and Naphthalene Oxidative Chemical Nucleases

Since the structural elucidation of duplex DNA, the construction of small molecules that recognise and react at specific sites to modify DNA structure, reactivity and biological repair mechanisms has been an area of considerable research interest. The discovery of the first synthetic chemical nuclease [Cu(phen) 2 ] 2+ (where phen = 1,10-phenanthroline) has sparked intensive effort toward the development of new artificial metallonucleases. [Cu(phen) 2 ] 2+ binds both nucleic acids and proteins without specificity inducing general toxicity, and is thus considered a “promiscuous” agent. Accordingly, manipulation of this chemotype represents an interesting developmental challenge. The first part of this work thus reports a range of novel Cu 2+ chemical nucleases of [Cu(phen)(N,N´)] 2+ carrying designer phenazine type-intercalators (where N,N´ = DPQ, DPPZ and DPPN) were developed to identify how systematic extension of the ligated phenanthrene group influences nucleotide binding affinity, base selectivity, oxidative chemical nuclease activity, and cytotoxicity within human cancer cells. Agents within this series showed potent intercalative selectivity with high-affinity binding constants among the highest reported to-date. Since the introduction of two metal centres into complex scaffolds has uncovered nucleic acid binding interactions not possible through the use of simple univalent compounds— with recent examples including the cytotoxic ‘self-activating’ DNA oxidant [Cu 2 (µ- terephthalate)(phen) 4 ] 2+ —the development of new polynuclear complexes is an area of considerable research interest. With this in mind, the second part of this thesis reports a new di-Cu 2+ complex [Cu 2 (tetra-(2-pyridyl)-naphthalene)Cl 4 ] (Cu 2 TPNap) that was rationally designed based on i.) the utility of a tetra-2-pyridine ligand scaffold for efficient nucleic acid catalytic cleavage within previously reported di-Zn 2+ systems, and ii.) the introduction of an aromatic DNA binding moiety that can potentially enhance both nucleic acid targeting and binding affinity. Using a range of molecular biological and spectroscopic techniques, Cu 2 TPNap was identified to bind DNA non-intercalatively at the major groove inducing guanine-cytosine specific deformation and condensation. The complex oxidatively damages DNA through a superoxide-mediated process leading to strand breakages in the absence of co-activating exogenous species. The final part of this thesis reports the discovery of a new class of DNA condensation agent featuring a C 3 -symmetric opioid scaffold. These agents, which may have potential gene transfection properties, were identified to collapse the tertiary structure of duplex DNA polymers through phosphate ionic interactions at a protonated piperidine site on the opioid molecule. Using a series of molecular biological and biophysical assays, the binding interactions and structural requirements for this new class of agent were uncovered and are described herein

Metallodrugs as inducers and inhibitors of chemical nuclease activity

Manipulation of DNA is both an intrinsic and essential component of molecular biology and biotechnology. Reagents capable of cutting DNA are applied within these fields as probes for DNA structure and function, with the ultimate aim being the design of target-specific—customised endonucleases—capable of modifying genomic DNA. Thus, DNA cleaving reagents are essential tools for both sequence analysis and genome engineering. Furthermore, the discovery of new molecular mechanisms by which small molecules modify DNA structure, reactivity, and biological repair contributes significantly to potential drug development. The chemical nuclease of [Cu(Phen)2]2+ (where Phen = 1,10-phenanthroline), is a well studied reagent which randomly cleaves nucleic acids in the presence of molecular oxygen (or hydrogen peroxide) upon reduction to Cu+. In addition, compounds based on this chemotype have found application in the biological field as antimicrobial and anticancer agents, DNA intercalators, and as nucleoside constituents for incorporation into the DNA backbone. [Cu(Phen)2]+ oxidises duplex DNA without specificity, predominately at the minor groove with C-H bonds at C1′, C4′, and C5′ being the main targets of hydrogen atom abstraction. The aim of this research was to extend structure-activity relationships of Cu2+-Phen complexes containing sterically functionalized pendant carboxylates and to investigate how synthetic extension of the ligated phenazine ligand within this complex model influences DNA recognition and oxidative degradation. These compounds have shown an enhanced DNA recognition relative to the well-studied chemical nuclease, [Cu(Phen)2]+. Furthermore, the effects of nuclearity on DNA oxidation were elucidated using the [Cu(-terephthalate)(Phen)4]2+ cation with results showing potent DNA oxidation in the absence of exogenous reductant. Many compounds developed in this work constitute a series of novel anticancer leads capable of intracellular DNA oxidation leading to genomic double strand breaks. In addition to the application of developmental metallodrugs as inducers of chemical nuclease activity, the effects of cytotoxic trinuclear platinum(II) complexes as high-affinity DNA binders that inhibit—or block—endonuclease enzyme recognition and excision are reported through a wide variety of biophysical and molecular biological methods

Structure, dynamics and hydration in drug-DNA recognition

The role of deoxyribonucleic acids in the cell has made DNA an attractive target for drug molecules. The anthracycline antitumour antibiotics are potent cytotoxic agents that have found widespread use in cancer chemotherapy. Nogalamycin binds DNA through intercalation, preferentially to 5'-TpG and 5'-CpG sites, by threading through the DNA helix and interacting with both the major and minor grooves simultaneously. In this thesis, the interaction of nogalamycin with the 5'-TpG site has been investigated using synthetic oligonucleotide duplexes and a combination of high-resolution NMR techniques and NOE-restrained molecular dynamics simulations. The solution structure of the 1: 1 complex with d(ATGCAT)2 is described with NOE data unambiguously identifying the position and orientation of the bound drug molecule, allowing conclusions to be drawn regarding the specificity for the TpG site. Binding at one TpG site sterically blocks the interaction at the symmetrically equivalent CpA site. The structural studies are extended to investigate by NMR the role of solvation in drug- DNA recognition and binding. Based on the sign and magnitude of solute-solvent NOEs, it is shown that only a small subset of water molecules visible in the crystal and MD structures are found to be bound in the solution complex, and that a number of these are involved in mediating drug-DNA interactions. The role of the dynamic network of water molecules in stabilising the complex in solution is discussed. Finally, the binding of nogalamycin at a TpG site carrying a DNA strand break has been investigated using a novel designed single-stranded intermolecular duplex consisting of two hairpins stabilised by GAA loops [d(ACGAAGTGCGAAGC)]. Although stacking of the two hairpins is weak, nogalamycin is shown to bind and stabilise a 1: 1 complex by binding at the intercalation site. The complex is discussed in terms of the mechanism by which nogalamycin is able to bind to premelted duplex DNA

Biophysical Characterization of the Binding of Homologous Anthraquinone Amides to DNA

The synthesis of four homologous anthraquinones (AQ I-IV) bearing increasing lengths of polyethylene glycol (PEG) side chains and their binding to AT- and GC-rich DNA hairpins are reported. The molecules were designed such that the cationic charge is at a constant position and the ethylene glycol units chosen to allow significant increases in size with minimal changes in hydrophobicity. The mode and affinity of binding were assessed using circular dichroism (CD), nuclear magnetic resonance (NMR), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC). The binding affinity decreased as the AQ chain length increased along the series with both AT- and GC-rich DNA. ITC measurements showed that the thermodynamic parameters of AQ I-IV binding to DNA exhibited significant enthalpy-entropy compensation. The enthalpy became more favorable while the entropy became less favorable. The correlation between enthalpy and entropy may involve not only the side chains, but also changes in the binding of water and associated counterions and hydrogen bonding. The interactions of AQ I-IV with GC-rich DNA have been studied via molecular dynamics (MD) simulations. The geometry, conformation, interactions, and hydration of the complexes were examined. As the side chain lengthened, binding to DNA reduced the conformational space, resulting in an increase in unfavorable entropy. Increased localization of the PEG side chain in the DNA groove, indicating some interaction of the side chain with DNA, also contributed unfavorably to the entropy. The changes in free energy of binding due to entropic considerations (-3.9 to -6.3 kcal/mol) of AQ I-IV were significant. The kinetics of a homologous series of anthraquinone threading intercalators, AQT I-IV with calf thymus DNA was studied using the stopped-flow. The threading mechanisms of the anthraquinones binding to DNA showed sensitivity to their side chain length. Fitting of the kinetic data led to our proposal of a two step mechanism for binding of AQT I, bearing the shortest side chain, and a three step mechanism for binding of the three longer homologs. Binding involves formation of an externally bound anthraquinone-DNA complex, followed by intercalation of the anthraquinone for AQT I-IV, then isomerization to another complex with similar thermodynamic stability for AQT II-IV