ANTHRAQUINONE BASED ANTICANCER DRUG MITOXANTRONE AND ITS COMPLEX WITH DNA

Abstract

Mitoxantrone belong to class of anthraquinone drugs, developed as an alternative to the apparently more cardiotoxic anthracycline antibiotics e.g. daunomycin and adriamycin. Mitoxantrone has shown promising perspective in patients with breast cancer, acute leukemia and non-Hodgkin's lymphoma. It affects DNA transcription as well as RNA processing. Several measurements reveal a preference for mitoxantrone binding to GC base pairs while in-vitro transcription assay has shown that the consensus sequences are 5'(A/T)CpA and 5'(A/T)CpG. It is of interest to understand the base specificity and structural factors that control chemical recognition process of mitoxantrone with DNA by difect independent evidence. Structural tools such as NMR spectroscopy and X-ray crystallography, coupled with molecular modeling techniques and theoretical studies have considerable impact in advancing our understanding of the structural selectivity and the molecular basis for drug-DNA interactions. We have used proton and phosphorus-31 Nuclear Magnetic Resonance spectroscopy followed by restrained MolecularDynamics simulations to study conformation of mitoxantrone-DNA complexes with DNA hexamer sequences, d-(TGATCA)2, d-(CGTACG)2 and d- (TGTACA)2. The Ph.D. thesis work has been reported in the form of seven chapters. Chapter 1 contains introduction of the subject, a comprehensive review of the literature and scope of thesis. Chapter 2 deals with the materials and methods used. The details of Nuclear Magnetic Resonance Spectroscopy methods used for the proton and phosphorus assignment are discussed. The strategies used for restrained molecular dynamics simulations and quantum chemical calculations are also discussed. Chapter 3 deals with quantum chemical calculations and restrained Molecular Dynamics simulation of Mitoxantrone. Structural and electronic properties of mitoxantrone have been studied using Density Functional Theory (DFT) employing B3LYP exchange correlation. The geometry of mitoxantrone is fully optimized without any constraints at the B3LYP/6-31G+(d,p) and B3LYP/6-31G(d,p) levels in vacuum, water andDMSO environments. Percentage variation of the difference between measured data and calculated results were 8.2 % and 4.6 % for 'H and 13C chemical shifts respectively. The calculated bond lengths and bond angles compare reasonably well with the experimental structure and the crystal structure data reported in literature. The geometrical parameters are relatively less influenced by the solvent effect but a noticeable change is seen in the values of the chemical shift due to inclusion of solvent. An overall analysis show that the B3LYP/6-31G+(d,p) level of theory predicts results which are in good agreement with the experiment. Chapter 4 to 6 deals with 31P and !H NMR and rMD studies on binding of mitoxantrone with DNA hexamer sequences d-(TGATCA)2> d-(CGTACG)2 and d- (TGTACA)2> respectively. The following experiments were performed on mitoxantrone- DNA complexes studied. lU and 31P NMR titration studies at various drug (D)/DNA duplex (N) ratios of upto 2.0 at 275 K and 298 K in 90% water and 10% D20. Temperature dependence of 31P and *H NMR of the mitoxantrone-d-(TGATCA)2 complex having D/N = 0.5, 1.0, 1.5 and 2.0 in the range of 275 - 328 K. 2D 31P - 31P exchange spectra of drug-DNA complex by aphase-sensitive NOESY using mixing time of 150ms and 200 ms at 275 Kfor D/N = 0.5, 1.0 and 2.0. 2D *H - *H NOESY at D/N = 0.5, 1.0, 1.5, 2.0 using mixing time xm = 100, 200, 300 ms at 275 Kin 90% H20 and 10% D20. Diffusion Ordered Spectroscopy (DOSY) experiments of complex of mitoxantrone-d-{TGATCA)2 and uncomplexed mitoxantrone in D20. Restrained molecular dynamics studies ofthe solution structure for the complexes ofmitoxantrone- DNA using inter-proton connectivity obtained from 2D NOESY as restraints. Binding of mitoxantrone with d-(TGATCA)2 shows, the ring protons of mitoxantrone 2H/3H, 6H/7H, and protons adjacent to aromatic ring i.e., 11NH, 11CH2 are shifted upfield up to 0.7 ppm due to binding. 31P NMR titration studies show absence of large downfield shifts in phosphate resonances. The TlpG2 resonance shifts downfield by -0.3 ppm at 278K. Intermolecular NOE connectivity observed between drug and DNA protons (2H/3H-T1CH3, 2H/3H-T1H6, 2H/3H-A6H8, 6H/7H-T1CH3, 6H/7HT1H2", 11CH2-T1CH3, 11CH2-A6H8, 11NH-T1CH3) show proximity of drug chromophore to terminal T1.A6 base pair. Conformational analysis of the final rMD structure shows DNA hexamer in complexed state adopts a conformation close to that of canonical B-DNA structure. These observations point towards external binding mode of mitoxantrone to d-(TGATCA)2. Preferably with mitoxantrone stacked between two molecules ofDNA. o 1 2D P NMR exchange spectrum of mitoxantrone complexed with d-(CGTACG)2 shows strong exchange correlation between bound and free ClpG2 resonance at stoichiometric ratio of 0.2, 0.5 and 1.0. The bound ClpG2 resonance is 0.40 ppm downfield shifted with respect to its free resonance. Absence of large downfield shift in 31P NMR spectra suggest that there is no characteristic unwinding of the DNA helix due to change in backbone torsional angle C, , C3'-03'-P-05' from gauche" to trans as observed with intercalators. There is a slow exchange on NMR time scale between free and bound species as exhibited by separate bound and free resonance for DNA protons - T3NH, G6NH, G2NH, T3CH3 and 11NH of mitoxantrone. Mitoxantrone protons 1lNHb, 10H/40H, 12CH2 / 13CH2 and 14CH2 are simultaneously close to CI and G6 residue protons, which are located on opposite sides of C1.G6 base pair. This is not possible if mitoxantrone aromatic chromophore intercalates between base pairs of DNA. The presence of intra base pair, sequential inter base pair and all sequential intramolecular NOE connectivities, show that all base pairs in d-(CGTACG)2 are intact ondrug binding. Mitoxantrone binds externally to d-(CGTACG)2 and the aromatic chromophore stacking with C1.G6 base pairs leading to large upfield shifts in 6H/7H, 2H/3H and particularly 11NH protons (A8~ 0.95 ppm upfield). On addition of mitoxantrone to d-<TGTACA)2, G2NH and T3NH resonances, gives rise to new set of broad signals upfield at the expense of the intensities of their free resonances. 2D 31P NMR exchange spectrum shows strong exchange correlation between bound and free TlpG2 resonance at stoichometric ratio of 0.5. The bound TlpG2 resonance is 1.15 ppm downfield shifted with respect to its free resonance. The significantly large downfield shift may be attributed to local drug-induced distortions at TlpG2 step leading to change in phosphate backbone conformation from gauche, gauche (a = -60°, C= -90°) to gauche, trans (a = -60°, £ = -180°). The existence of bound and free resonance for G2NH, T3NH and 11NH (drug) clearly demonstrates that the drug indeed binds to the DNA hexamer and there is exchange of free and bound DNA on NMR time scale at 275 K. Several intermolecular contacts observed are close to T1.A6 base pair. HNHb gives close contacts with T1CH3, T1H6, A6H2, A6H8, T1H1' and A6H1'. 2H/3H is close to A6H8 as well as with T1H1' and T1H4'. Similarly 12/13CH2 gives close contacts with T1CH3, A6H8 and T1H1'. Such NOE connectivities are possible only if mitoxantrone aromatic chromophore binds externally to the terminal base pairs ofDNA. The aromatic chromophore ofmitoxantrone stacks with T1.A6 base pairs leading to large upfield shifts in 6H/7H, 2H/3H and particularly 11NH protons (A5 ~ 0.78 ppm upfield). Chapter 7 compares the binding of mitoxantrone with d-(TGATCA)2> d- (CGTACG)2 and d-(TGTACA)2 and its implications in understanding molecular basis of action of mitoxantrone.