INTERACTION OF LUTEOLIN AND MITOXANTRONE WITH G-QUADRUPLEX DNA SEQUENCES

Abstract

Telomeres are nucleoprotein complexes at the termini of linear chromosomes, which protect chromosomes from fusion and degradation. The extreme single stranded 3' end of the telomere comprises of simple tandem repeats of guanine-rich sequences (TTAGGG)n in humans and other vertebrates, with the potential to adopt quadruplex fold. G-rich sequences are also found in other biologically significant regions of the genome such as immunoglobulin switch regions, gene promoter regions of important proto-oncogenes, 5' untranslated regions (UTRs) of many mRNAs and other sequences associated with human diseases. Telomeric DNA attrition on cell replication ultimately leads to cell senescence, although in the majority of cancers, telomeres are maintained by the enzyme telomerase, which is expressed in greatly enhanced levels in these cells. The action of telomerase is inhibited on folding of its single stranded DNA substrate into a quadruplex. One prominent strategy that emerged in last decade for telomerase inhibition is the stabilization of G-quadruplex structures of telomeric DNA. The G-quadruplex formation and stabilization is facilitated by small molecules via displacement of telomeric end capping protein hPOT 1, uncapping of telomere and subsequent initiation of DNA damage response-mediated cell death. The G-quadruplex interactive ligands like trisubstituted acridine compound BRACO-19, RHPS4 , telomestatin, TMPyP4 and fluoro quinone derivative Quarfloxin, etc. exhibit anti cancerous activity in in-vivo tumor xenograft models. These ligands with their planar aromatic ring system stack effectively on planar G-tetrad via π-π interaction inducing replicative senescence in cancerous cells. However synthetic ligands exhibit toxic side effects such as cardiotoxicity, nausea, vomiting, etc. limiting their further application. This opens a window for compounds with similar efficacy but less toxicity. In this regard flavonoids, dietary plant derived polyphenols with extended aromatic ring system and biological properties viz. anti-oxidant, anti-cancer, anti-inflammatory and kinase inhibition are expected to be an ideal choice. They may possess activity comparable to their synthetic counterparts, with no apparent side effects and toxicity to normal cells, and hence offer an opportunity for more successful treatment of cancer. In the present study, We have shown the interaction of flavonoid, luteolin and mitoxantrone (MTX), a well know chemotherapeutic synthetic anthraquinone derivative with human telomere single repeat sequence d-(TTAGGGT) and Tetrahymena Sequence d-(TTGGGGT), which form right handed parallel stranded G-quadruplexes in presence of K+ ion with anti glycosidic torsional angles. The Ph. D. thesis work has been reported in the form of five chapters. xxii Chapter 1 reviews the literature on broad subject area of telomeres, telomerase enzyme, the strategies involved in telomerase inhibition and G-quadruplex interactive ligands. It also deals with biophysical and biochemical aspects of luteolin and mitoxantrone. Chapter 2 deals with the materials and methods used in the present work. Binding mode and stoichiometry of ligand-G-quadruplex complexes are determined by absorption and fluorescence spectroscopy. The conformational changes due to binding are monitored by Circular Dichroism (CD) measurements. Structural elucidation of ligand-G-quadruplex complex is done using Nuclear Magnetic Resonance (NMR) techniques followed by restrained Molecular Dynamics (rMD) simulations. The results have been validated with a cell based Telomere Repeat Amplification Protocol (TRAP) assay. Chapter 3 describes the exact binding mode, stoichiometry and conformational changes involved in mitoxantrone complexed with parallel G-quadruplex sequence d-(TTAGGGT)4. The UV-visible absorption spectrum of uncomplexed MTX shows four distinct bands at λmax 242, 276, 609 and 659 nm. We monitored the changes in the 662 nm peak as it is more sensitive to the change in mitoxantrone concentration and DNA. Upon addition of quadruplex to MTX, the absorbance maxima of both 659 and 609 nm peaks show hypochromicity accompanied by red shift of 15 and 11 nm, respectively. Presence of clear isobestic point at 677 nm indicates existence of single mode of interaction with a binding constant 2.23 x 105 M-1. Fluorescence intensity decreases on binding and is accompanied by 12 nm red shift, yielding binding stoichiometry (n) ~1.7. This is in agreement with the data obtained by Job plot method of continuous variation analysis, which gives inflection point at 0.66. The binding leads to thermal stabilization of G-quadruplex with an increase in Tm of 13◦C. Chapter 4 describes structural studies of MTX complexed with d-(TTAGGGT)4. Both one dimensional 1H and 31P NMR and two dimensional experiments viz COSY, NOESY, 1H-13C HSQC, 1H-31P HMBC have been used for assignment of various resonances in uncomplexed and complexed states. MTX is titrated with d-(TTAGGGT)4 at various Drug (D) to Nucleic acid (N) ratios up to 2.0 (D/N = 0.25 to 2.0) at different temperatures in the range of 278-318 K. Addition of MTX to d-(TTAGGGT)4 did not induce significant chemical shift variation of phosphate and proton signals. A maximum upfield shift of 0.06 ppm is observed in all NH protons of quadruplex on binding while MTX protons 2/3 H, 1/4 OH and 11 CH2 shift xxiii upfield by 0.53 ppm, 0.48 ppm and 0.31 ppm, respectively. Presence of imino signals resonating between 10.0 and 11.5 ppm is indicative of formation of G-quadruplex structure. NH signals of uncomplexed d-(TTAGGGT)4 disappear at 60◦C while that of complexed DNA disappeared at 85◦C, indicating stabilization of G-quadruplex on binding to MTX. NOESY spectra recorded with variable mixing times τm 100, 200, 250 ms are analysed at D/N = 1:1 and D/N = 2:1. The presence of all sequential connectivities between base-H1‘/H2‘, H2‖ and NH-NH protons, indicate that all G-quartets are intact. Intermolecular NOE connectivities between MTX protons 11 NH, 1/4 OH, 6/7 H and 12 CH2 are observed with sugar (H1', H2'/H2'', H4', H5'/H5'') and CH3 protons of T1, T2, and T7 bases of DNA. Absence of intermolecular NOEs between 11 NH-2/3 H, 11 CH2-2/3 H, 6/7 H-2/3 H reveal that MTX does not exist as dimer in complex, instead two molecules of MTX (one at T1pT2 step and second at G6pT7 step) bind to one molecule of d-(TTAGGGT)4 in the groove. Absence of significant downfield shift of 31P resonances (> 1.5 ppm) excludes the possibility of opening of base pair at any step to permit intercalative mode of binding. The observed NOE restraints have been used to build the structure of complex. Chapter 5 deals with the structural studies of luteolin complexed with two G-quadruplex sequences d-(TTAGGGT)4 and d-(TTGGGGT)4, which occur in human and Tetrahymena telomeres, respectively and differ at third base position. On titrating luteolin with DNA up to D/N ratio 1.0 no significant change in chemical shift of d-(TTAGGGT)4 protons is observed. However d-(TTGGGGT)4 protons show shift in G6 NH, T7 H6, and T7 CH3 protons on binding with luteolin accompanied by line broadening of imino signals. Aromatic protons H2', H6', H6, H8 and H3 of luteolin are shifted upfield on binding. All sequential connectivities, base- H1', H2'/H2''and NH-NH are found to exist. Imino signals resonate between 10.0-11.5 ppm indicating that DNA remains in G-quadruplex form on complexation. G-quadruplex structure is stabilized on binding as the NH signals are found to persist at 80 ◦C, which disappear in uncomplexed DNA at 55 ◦C. Most of the intermolecular NOE contacts appeared between H2', H6', H6, H8 protons of luteolin and H1', H2'/H2'', CH3 and NH of T1/T2/G6/T7 bases of d-(TTAGGGT)4. In case of luteolin-d-(TTGGGGT)4 complex, same luteolin protons are involved in making short contacts with G5H1', G6H1', G6H2'', G6 NH and T7H1' protons. The phosphate resonances of d-(TTAGGGT)4 do not show any significant shift while that of d-(TTGGGGT)4 show downfield shift of 0.12 ppm at G6pT7 step on binding. This is indicative of preferential binding of luteolin at G6pT7 step in case of d-(TTGGGGT)4 whereas in d-(TTAGGGT)4, additional binding site at T1pT7 exists. xxiv Hence we conclude that both MTX as well as luteolin interact with G-quadruplex DNA. The results of TRAP assay confirm dose dependent telomerase inhibition by these ligands, which can be promising G4 ligands with implications towards G-quadruplex mediated telomerase inhibition for anticancer therapy.