STRUCTURAL AND BIOPHYSICAL STUDIES OF INTERACTION OF ANTHRACYCLINES WITH G-QUADRUPLEX DNA

Abstract

Guanine rich sequences form non-canonical four stranded G-quadruplex DNA structures, which are self assembled array of guanine tetrads formed through Hoogsteen base pairing stabilized by the presence of central monovalent cationic tract. Telomerase, a ribonucleoprotein enzyme, elongates linear DNA at the end of chromosome which forms a dynamic nucleoprotein assembly called telomere. Telomeric regions in humans and other organisms comprise guanine-rich repeats of few hundred base pairs in 3´-end overhangs which can fold into G-quadruplex DNA (G4) structure under in vivo conditions. Absence of telomerase activity leads to end to end fusion of linear chromosomal DNA ends and subsequent cell senescence through apoptosis signaling due to chromosomal instability. Tumor cells become immortalized through activation of telomerase enzyme that stabilizes the length of telomeres. Telomerase levels have been found to correlate with cancer progression and metastatic state and the enzyme is not expressed in normal human tissue but is present in at least 85% of tumor cells. The RNA template of telomerase as well as capping function require extended single stranded DNA primer for effective hybridization and folding of telomeric repeats into higher order DNA structures (e.g. G-quadruplex) would hinder these processes. The general consensus is that G-quadruplex binders, which stabilize G4 structure, interfere with DNA damage response activation, oncogene expression and genomic stability and hence possess potential to act as regulatory elements of different processes. Consequently G-quadruplex binding agents can serve as a viable therapeutic strategy, also due to their selectivity since they would not show cytotoxic effect outside tumor. This paved the way for discovery of novel anticancer agents by focusing much research activity on design approaches based on molecular interactions of ligands specific with G4 DNA sequences. A large number of compounds e.g. alkaloids, flavonoids, porphyrins, anthraquinones, anthracyclines, porphyrin derivatives, metalloporphyrins, etc. are being evaluated for their affinity and specificity for G4 DNA. Anthracyclines have been used in past as anticancer drugs. Daunomycin isolated from Streptomyces peucetius is found to be active against acute lymphoblastic or myeloblastic leukemias, while its 14-hydroxy derivative, adriamycin (doxorubicin) is active against specific solid tumors. It is well established that these drugs interact with duplex DNA through classical mode of intercalation between base pairs by uncoiling DNA, block DNA/RNA synthesis and act as topoisomerase-II poison. Recent findings point towards the role of daunomycin/adriamycin in maintenance of telomeres and induced telomere dysfunction by suppressing telomerase association with telomeres. Competition dialysis and spectroscopy techniques have revealed that the anthracyclines bind to different forms of DNA and exert their VI influence by following multiple strategies involving complex mechanisms, which are not well understood. In the present thesis, a comprehensive study of interaction of three anthracycline based ligands namely, daunomycin, 4’-epiadriamycin and adriamycin, with 7-mer tetramolecular parallel stranded [d-(TTGGGGT)]4 and 22-mer unimolecular d-[GGGG(TTGGGG)3] G4 DNA, containing telomeric DNA sequence TTGGGG from protozoan Tetrahymena thermophilia, has been undertaken by absorption, steady state & life time fluorescence, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy. We have also determined real time binding affinity using Surface Plasmon Resonance (SPR). The melting profiles of DNA and its complexes with these ligands have been obtained by absorption, CD and Differential Scanning Calorimetry (DSC). These investigations have been supplemented by molecular docking studies to understand mode of interaction. The thesis has been divided into eight chapters. Chapter 1 comprises general introduction on various forms of DNA, telomere, telomerase and structure of G-quadruplex DNA. A detailed literature survey on various ligands, particularly anthracyclines, interacting with G-quadruplex; study of ligand-quadruplex DNA interactions by various biophysical techniques; structure of their complexes determined by NMR techniques; the implications of telomerase inhibition as a strategy for development of anticancer therapeutics; and biological evidence of telomerase dysfunction by anthracyclines are discussed. The specific objectives of the present research work have been spelled out. Chapter 2 comprises materials and methods used for carrying out the structural and biophysical studies on interaction of the three ligands with the two selected G-quadruplex DNA sequences. The mathematical models and equations used to obtain binding parameters like binding constant, quenching constant, stoichiometry of ligand-DNA complexes, etc. using biophysical studies such as - UV-visible absorption, steady state and time-resolved Fluorescence and Circular Dichroism (CD) spectra are stated. Techniques of determining binding affinity by Surface Plasmon Resonance (SPR) and thermal denaturation by Differential Scanning Calorimetry (DSC) are discussed. The detailed experimental parameters used in 1D 1H, 31P and 2D Nuclear Magnetic Resonance (NMR) experiments namely, 1H-1H NOESY at different mixing times, 1H-13C HSQC, 1H-31P HMBC and their pulse programs are given. The methodology used for building NMR based model of ligand-DNA complex following restrained Molecular Dynamics (rMD) and molecular docking are also stated. VII Chapter 3 presents studies of daunomycin binding to 7-mer [d-(TTGGGGT)]4 G4 DNA by absorption, fluorescence, and CD spectroscopy. Also molecular docking and thermal denaturation by absorption/CD spectroscopy are given. Absorbance, flourescence and CD spectra show significant change on interaction with no change in wavelength maxima. The binding affinity is Kb ~105 M-1 for the 1:1 and 2:1 stoichoimetric drug-DNA complexes. CD spectra show minor changes in DNA conformation suggesting end stacking and groove binding of daunomycin monomer. The daunomycin dimers present in free state in solution, are disrupted on binding. Thermal stabilization of G4 DNA by 10-15 °C upon daunomycin binding is obtained from absorbance and CD measurements. The molecular docking studies establish well defined binding of daunomycin at two different sites of G4 DNA. Chapter 4 comprises results of binding studies of daunomycin complexation to 7-mer [d-(TTGGGGT)]4 G4 DNA, primarily by NMR techniques, SPR and Diffusion Ordered SpectroscopY (DOSY). Proton and phosphorus-31 NMR spectra show chemical shift changes, line broadening and sequence specific interaction with a clear proof of absence of intercalation of daunomycin chromophore between base quartets or stacking between G-quadruplexes. Several Nuclear Overhauser Enhancement (NOE) correlations between daunomycin and G4 DNA are found. Restrained Molecular Dynamics (rMD) simulations using short inter proton distance contacts depict interaction at molecular level. The interactions involving ring A and daunosamine protons, stacking of aromatic ring of daunomycin with terminal G6 quartet by displacing T7 base and external groove binding close to T1-T2 bases lead to thermal stabilization of 15 °C, determined by DSC experiments. Chapter 5 presents results on interaction of daunomycin with 22-mer d-[GGGG(TTGGGG)3] G4 DNA in K+ rich aqueous solution. Using SPR, we demonstrate real time binding. Changes in absorption/CD spectra and efficient quenching of fluorescence accompanied by minor change in wavelength establish external binding of daunomycin with no scope of classical intercalation as observed on its binding to duplex DNA. Multiple stoichiometric complexes coexist in solution. Proton NMR spectra show significant shifts in aromatic protons of ring B/D, daunosamine sugar protons and 14 short inter molecular contacts, exhibiting specificity of interaction. Large downfield shifts in phosphorus-31 NMR spectra are absent. Molecular docking confirms external binding by formation of complex with negative binding energy. DSC experiments show binding profiles with melting temperature Tm increasing with incremental addition of daunomycin to DNA and total thermal stabilization, ΔTm = 10 °C. VIII Chapter 6 comprises absorption, fluorescence and CD studies on binding of 4’epiadriamycin and adriamycin with 7-mer [d-(TTGGGGT)]4 G4 DNA. Absorbance measurements show 56% hyposchromism while 95% quenching of fluorescence of ligands is observed with no change in wavelength maxima. The binding affinity is Kb ~105-106 M-1. Fluorescence lifetime remains unaffected on complexation. CD spectra show significant changes and ligands are found to bind in monomeric form. Thermal stabilization of G4 DNA by 13-16 °C is determined by absorbance and CD measurements. The molecular docking studies show binding of ligands at two different sites of G4 DNA. Chapter 7 comprises extensive analysis of binding of 4’epiadriamycin and adriamycin with 7 mer [d-(TTGGGGT)]4 G4 DNA by NMR techniques. SPR and Diffusion Ordered SpectroscopY (DOSY) experiments are also carried out. Proton and phosphorus-31 NMR spectra show chemical shift changes, line broadening and sequence specific interaction with a clear proof of absence of intercalation of daunomycin chromophore between base quartets or stacking between two G4 DNA structures. Significant difference in phophrus-31 NMR and inter proton ligand-DNA distances are observed in the complexes formed by 4’epiadriamycin and adriamycin with G4 DNA. Restrained molecular dynamics simulations have been performed to get optimised conformation of complexes. The interactions at molecular level lead to thermal stabilization up to 23 °C, determined by DSC experiments. Chapter 8 comprises absorption, fluorescence and CD studies on binding of 4’epiadriamycin and adriamycin with 22-mer d-[GGGG(TTGGGG)3] G4 DNA in K+ rich aqueous solution. SPR results demonstrate real time binding. Changes in absorption/CD spectra and efficient quenching of fluorescence are accompanied by minor change in wavelength. Isobestic or iso-emissive points are not observed. Job plot shows that 1:1 and 2:1 ligand-G4 DNA stoichiometric complexes coexist in solution. Molecular docking confirms external binding by formation of daunomycin-DNA complex with negative binding energy -6.0 to -7.7 kcal/mole. DSC experiments show binding profiles with melting temperature Tm increasing with daunomycin to DNA ratio and total thermal stabilization, ΔTm = 5 °C. The findings have implication in design of analogues that could produce de novo anthracycline that acts as a potent telomerase inhibitor with enhanced selectivity towards G-quadruplex and hence reduced cellular toxicity.