INTERACTION OF FLAVONOIDS AND MITOXANTRONE WITH TETRAHYMENA G-QUADRUPLEX DNA SEQUENCE

Abstract

Telomeres are nucleoprotein regions found in the terminal ends of eukaryotic chromosomes. Their function is to stabilize and protect the ends of the chromosomes from degradation. In almost all eukaryotes, the telomeric repeat contains runs of Guanine bases. Some examples include the ciliates Tetrahymena, TTGGGG, and Oxytricha nova, TTTTGGGG; the plant, Arabidopsis, TTTAGGG; and the vertebrate repeat, TTAGGG. This G-rich telomeric overhang readily forms G-quadruplex structures; the basic unit of which is the G-quartet, a planar array of four guanines. DNA structures stabilized by G-quartets are variously referred to as G-quadruplex, G-tetraplex or G4 DNA. With each cell division some of the DNA is lost from the ends of chromosomes (telomere region) due to end replication problem. When telomeres reach a critical minimum length, cells cannot divide and cellular senescence and apoptosis is induced. Telomerase is a ribonucleoprotein complex, which preserves telomere length in stem cells, germ cells, and cancer cells by adding hexameric (TTAGGG) repeats to the ends of chromosomes. As stem cells, germ cells, cancer cells express the telomerase activity; adult somatic cells lack this enzyme. About 90% of cancer cells contain short telomeres, but exhibit high telomerase activity. Hence telomerase inhibition is a strategy to prevent cancer development and progression. There are several strategies to inhibit the activity of telomerase enzyme; an important one among them is stabilization of G-Quadruplex DNA structures, which act as substrate for the telomerase enzyme. Majority of the known G-quadruplex stabilizing ligands have an extended planar aromatic ring system and stack effectively on planar G-tetrad via π-π interaction. In the present work we have carried out studies on the interaction of anticancer drug Mitoxantrone (MTX), flavonoids, Rutin and Quercetin, with Tetrahymena G-quadruplex DNA sequence which forms a tetramolecular parallel structure, by various biophysical techniques. Structures of these three compounds complexed to G-quadruplex d-(TTGGGGT)4 have been determined using 2D NMR techniques followed by restrained Molecular Dynamics (rMD) simulations. The biological activity of these three compounds have been determined by 1-(4, 5-Dimethylthiazol-2-yl)-3, 5- diphenyl formazan (MTT) assay, Telomere Repeat Amplification Protocol (TRAP) assay. The thesis is divided into six chapters. Chapter 1 gives the introduction about telomeres in general, G-quadruplex structures, telomerase and its relationship to cancer. It also explains the strategies involved in the inhibition of telomerase enzyme, in particular, by stabilizing the G-quadruplex vi structures. The detailed literature survey of structural and functional aspects of ligands used to stabilize the G4 DNA structures is also discussed. Chapter 2 gives the detailed materials and methodologies used in the present work, that is, UVVisible Absorption, Fluorescence and Circular Dichroism (CD) spectroscopy as well as Surface Plasmon Resonance (SPR). The pulse programs of one and two dimensional Nuclear Magnetic Resonance (NMR) spectroscopy experiments and the steps for restrained Molecular Dynamics (rMD) simulations are also described. The procedure used to calculate binding constants and stoichiometry from the spectroscopic data, protocols for the, MTT, TRAP, PCR stop assays are also given in this chapter. Chapter 3 describes the spectroscopic studies of interaction of flavonoids rutin, quercetin and mitoxantrone with G-quadruplex sequence d-(TTGGGGT)4. Addition of increasing amounts of d- (TTGGGGT)4 to MTX leads to the change in the absorbance spectra of the MTX molecule. The absorbance maximum of 659 nm of monomer MTX peak shifts towards the longer wavelength, 674 nm, upon binding to quadruplex DNA. At higher D/N ratios, the absorption maxima also show 50 % hypochromism. The Fluorescence emission spectra shows red shift of 12 nm, binding constant of 4.1x105 M-1 and number of binding sites n = 2.4 . Job plot method of continuous variation analysis gives the inflection point at 0.66 and at 0.8 confirming thereby that two drug molecules bind to DNA. The CD results show that quadruplex structure is stabilized upon interaction with MTX. The Surface Plasmon Resonance (SPR) experiments yield dissociation constant of 8.75x10-5 M for the interaction of MTX with d-(TTGGGGT)4. Chapter 4 describes the detailed NMR study of binding of MTX with G-quadruplex DNA sequence d-(TTGGGGT)4. A combination of both one dimensional 1H and 31P experiments along with two dimensional NOESY (τm = 100, 200, 250 ms), COSY, 1H- 13C HSQC, 1H- 31P HMBC experiments has been used to assign resonances of uncomplexed and complexed d-(TTGGGGT)4 with MTX. The titration of MTX to quadruplex DNA d-(TTGGGGT)4 results in the broadening of the proton resonances of T7 H6, G6 H8 and NH resonances upto D/N ratio 1.0, which get sharpened on further increase of D/N ratio to 2.0. The chemical shift data show upfield shift ~ 0.14-0.23 ppm in all G NH resonances. The aromatic ring proton 2/3H of MTX shifts upfield by 0.4 ppm, while alkylamine side chain protons of 13 CH2 and 14 CH2 shift by 0.11 ppm. Analysis of the NOESY spectra of MTX-d- (TTGGGGT)4 complex at D/N ratio 2.0 shows existence of 35 intermolecular NOE cross peaks vii between drug and DNA protons and 14 intra/inter molecular cross peaks within drug protons. The observed intermolecular peaks show proximity of MTX with terminal bases T1, T2 and G6, T7. The melting experiments show that G3 and G6 NH resonances disappear at 338 K in uncomplexed d- (TTGGGGT)4, whereas in 2:1 complex, the G3 and G6 NH resonances are visible even at 363 K. The 31P experiments shift upfiled by 0.146 and 0.125 ppm at G5pG6 and G6pT7 steps, respectively. The observed NOE restraints have been used to get the structure of complex. Chapter 5 describes the NMR study of interaction of flavonoid quercetin with quadruplex DNA sequence d-(TTGGGGT)4. Titration of quercetin with d-(TTGGGGT)4 results in upfield shift of proton resonances. The NH resonances of all G tetrad forming Guanine residues show small upfield shift by 0.02 ppm. The melting studies show that d-(TTGGGGT)4 gets stabilized upon complexation with quercetin. The intermolecular NOEs are observed between aromatic H2’ and H6’ of quercetin with T2 and G3 residues. The observed NOE restraints have been used to build the model of complex. Chapter 6 describes the detailed NMR study of binding of flavonoid rutin with G-quadruplex DNA sequence d-(TTGGGGT)4. Titration of rutin to d-(TTGGGGT)4 G-quadruplex sequence results in shift of quadruplex resonances; all G NH proton resonances shift upfield, G6 NH shows maximum shift of 0.15 ppm and T7H6 shifts downfield by 0.20 ppm. The results of 31P experiments suggest binding of rutin at G6pT7 step, which shows maximum downfield shift of 0.21 ppm. The two dimensional NOESY spectra show eight intermolecular cross peaks in 1:1 ruitn-d-(TTGGGGT)4 complex, all of them being with G6 and T7 bases. The important intermolecular contacts are H6, H8, H6’, H2 of rutin with G6 NH; H6’ and H2' of rutin with G6 H1'. All rutin proton resonances shift upfield, maximum shift of 0.69 ppm observed for H6 proton. The melting studies show that d- (TTGGGGT)4 gets stabilized upon complexation with rutin. In conclusion, the present study shows that anticancer drug MTX, and naturally occurring flavonoids, rutin and quercetin interact with d-(TTGGGGT)4 quadruplex sequence and stabilize it. Results of TRAP assay show, dose dependent inhibition of telomerase enzyme. The present results have major implications on the understanding of binding mode of G-quadruplex ligands and development of effective anticancer agents based on the strategy of stabilizing of G-quadruplex DNA sequence, there by inhibiting telomerase enzyme.