INTERACTION OF ANTHRACYCLINES WITH HUMAN TELOMERIC G-QUADRUPLEX DNA

Abstract

Anthracyclines derived from Streptomyces peucetius, rank among the most effective chemotherapeutic agents. Adriamycin and its 4’-epi-isomer, epirubicin are found to be effective anticancer agent. Both comprise an aromatic chromophore (ring BCD), a cyclohexyl moiety (ring A) to which a sugar group is attached at C7 position but different orientation of OH residue at C4’ position of daunosamine sugar. Epirubicin causes lesser cardiotoxicity. They interfere with functioning of DNA topoisomerase-II by inhibiting religation of DNA and enhancing DNA strand breaks by formation of drug-DNA-enzyme ternary complex. Structural modifications in the sugar moiety have been envisaged as a key to improving the anticancer effectiveness of anthracyclines. Recent reports indicate influence of anthracycline on telomere maintenance. Telomeric regions in humans comprises guanine (G)-rich repeats of few hundred bases at 3´-end, which can fold into G-quadruplex DNA (G4) structure under in vivo conditions. The G4 DNA is a non canonical tetraplex structure having 4 guanines in a plane held by Hoogsteen hydrogen binding in the presence of K+/Na+ cations. Telomerase, a ribonucleoprotein enzyme, correlate with cancer progression/metastatic state as the enzyme is not expressed in normal human tissue but is present in at least 85% of tumor cells. Tumor cells become immortalized through activation of telomerase enzyme. The RNA template and capping function require extended single stranded DNA primer for effective hybridization. Folding of G-rich repeats into higher order G4 DNA structures has been visualized in mammalian cells and is found to hinder these processes. The stabilization of G-quadruplex by binding to small molecules interferes with DNA damage response activation, oncogene expression and genomic stability and hence acts as regulatory elements of different processes. Consequently G-quadruplex binding agents can serve as a viable therapeutic strategy due to their selectivity since they would not show cytotoxic effect outside tumor. Competition dialysis and spectroscopy techniques have revealed that anthracyclines bind to different forms of DNA, cause telomere dysfunction and exert their influence by following multiple strategies involving complex mechanisms, which are not well understood. In the present thesis, a comprehensive study of interaction of two anthracycline based ligands namely, adriamycin and epirubicin, with 7-mer tetra-molecular parallel stranded [d (TTAGGGT)]4 and 22-mer intramolecular d-[AGGG(TTAGGG)3] G4 DNA, containing human V telomeric DNA sequence TTAGGG, has been undertaken by absorption, steady state & life time fluorescence, Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy. The investigations have been supplemented by molecular docking studies. Real time binding affinity has been determined using Surface Plasmon Resonance (SPR). The melting profiles of DNA and its complexes with ligands have been obtained by CD and Differential Scanning Calorimetry (DSC). Conformation of ligand-[d-(TTAGGGT)]4 complex has been obtained by following restrained Molecular Dynamics (rMD) simulations using torsional angle and distance constraints from two dimensional NMR experiments. The thesis is divided into seven chapters. Chapter 1 comprises introduction on basic structure of DNA, different forms of DNA, telomeric DNA, telomerase enzyme and its cellular function. Literature survey on ligands binding to DNA, the ligand-quadruplex DNA interactions by various biophysical methods, structure of ligand-G4 DNA complexes determined by X-ray/NMR techniques, evidence of disruption of inter play between telomerase and telomere causing telomere dysfunction by anthracyclines, etc. has been discussed. The scope of present research work along with specific objectives has been mentioned. Chapter 2 comprises materials and methods used for carrying out biophysical and structural studies on interaction of ligands with selected G4 DNA sequences. The mathematical models for fitting of experimental data and equations used to obtain binding parameters like affinity constant, quenching constant, stoichiometry of ligand-DNA complexes, etc. from uv-visible absorption, steady state and time-resolved fluorescence and CD spectra are given. SPR and DSC techniques for determining binding affinity and thermal melting profiles, respectively 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 stated. The methodology used for building NMR based model of ligand-DNA complex following restrained Molecular Dynamics (rMD) simulations and molecular docking using AutoDock are also stated. Chapter 3 presents studies of adriamycin binding to 7-mer [d-(TTAGGGT)]4 G4 DNA by absorption, fluorescence, and CD spectroscopy and thermal denaturation by CD/DSC. Absorbance and flourescence spectra show 57% and 95% change on interaction accompanied VI by red shift in wavelength maxima by 8-15 nm, respectively and change in slope of their variation with ligand (Drug) to G4 Nucleic acid (N) molar equivalent ratio at D/N = 0.5, 1 and 2. The binding affinity Kb1 = 9.8x105 M-1 and Kb2 = 6.7x105 M-1 at two independent sites is found to be higher than that reported for daunomycin earlier. The magnitude of CD bands change by 57-70%. The adriamycin dimers present in free state in solution, are disrupted on binding. Thermal stabilization of G4 DNA ~12.5-28.1 °C upon binding is obtained from CD/ DSC measurements. NMR spectra shows, line broadening and chemical shift changes. The results show external binding at two different sites of G4 DNA. Chapter 4 comprises results of adriamycin complexation to 7-mer [d-(TTAGGGT)]4 G4 DNA, primarily by NMR techniques. 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 adriamycin chromophore between base quartets or stacking between G quadruplexes due to presence of sequential NOEs and absence of large downfield shifts in 31P NMR spectra. Thymine imino protons are visible in proton NMR spectra at 5 °C due to their immobilization on binding. Restrained Molecular Dynamics (rMD) simulations using short inter proton distance contacts, including 15 Nuclear Overhauser Enhancement (NOE) correlations between adriamycin and G4 DNA protons, depict interaction involving ring D aromatic and methoxy protons, partial stacking of aromatic ring of adriamycin with terminal G6 quartet by displacing T7 base and external groove binding close to T1-T2-A3 bases leading to thermal stabilization ~ 24 °C. Chapter 5 comprises absorption, fluorescence and CD studies on binding of epirubicin with 7 mer [d-(TTAGGGT)]4 G4 DNA. Absorbance and flourescence spectra show 57% hypochromism and 95% quenching on interaction accompanied by red shift in wavelength ~7 nm, respectively and change in slope of their variation with D/N at D/N = 0.5, 1 and 2. The overall results show that epirubicin binds to G-quadruplex DNA with affinity, Kb1 = 3.8x106 M 1 and Kb2 = 2.7x106 M-1, at two independent sites externally. Fluorescence lifetime remains unaffected on complexation. CD spectra show significant changes and ligands are found to bind in monomeric form. The interactions induce thermal stabilization of DNA by 13.2-26.3 °C. Binding in presence of lower K+ concentration and to antiparallel form of G4 DNA formed in presence of 100 mM Na+ is found to have lower affinity. NMR spectra shows, changes in chemical shift and line broadening. VII Chapter 6 comprises extensive analysis of binding of epirubicin with 7-mer [d-(TTAGGGT)]4 G4 DNA by NMR techniques. The upfield shift in ring D protons is significantly large and decrease in order 2H (0.66 ppm) > 3H (0.55 ppm) > 1H (0.48 ppm) > 4OCH3 (0.39 ppm). The upfield shift in daunosamine sugar proton is up to ~0.25 ppm. G6NH is severely broadened and shifts maximum upfield ~0.23 ppm as compared to G4/G5NH (~0.09 ppm). Restrained Molecular Dynamics (rMD) simulations using short inter proton distance contacts, including 09 Nuclear Overhauser Enhancement (NOE) correlations between epirubicin and G4 DNA protons, show interaction at molecular level between ring D aromatic/methoxy protons and T1, T2, A3 and G6 base protons. The interaction leads to thermal stabilization ~16 °C. Chapter 7 presents results on interaction of adriamycin and epirubicin with 22-mer d [AGGG(TTAGGG)3] G4 DNA in K+ and Na+ rich aqueous solution by SPR, absorption, fluorescence, CD and thermal denaturation. Epirubicin and adriamycin bind with affinity, Kb, = 2.5x105 and 5.2x105 M-1, respectively in monomeric form leading to hypochromism, fluorescence quenching and ellipticity changes without any significant shift in λmax and binding induces thermal stabilization ~13.0 and 11.6 °C, respectively in K+ rich solution. Presence of Na+ ions did not induce any thermal stabilization. Molecular docking confirmed external binding at grooves and loops of G4 DNA involving 4OCH3 of ring D, 9COCH2OH of ring A, 4’OH/H and 3’NH3+ of daunosamine sugar. The role of 4’OH group of daunosamine sugar on binding is discussed. Thermal stabilization induced by specific ligand-G4 DNA interactions is likely to hamper telomere association with telomerase enzyme and contribute to drug-induced apoptosis in cancer cell lines besides causing damage to duplex DNA. The findings pave the way for drug designing in view of immense possibilities of altering substituent groups on anthracyclines for enhancement of efficacy, reduced cell toxicity as well as using alternate mechanism of its interaction with G4 DNA, causing interference in telomere maintenance pathway by inducing telomere dysfunction.