Ritu Barthwal

Interaction of anticancer drug mitoxantrone with DNA hexamer sequence d-(CTCGAG)2 by absorption, fluorescence and circular dichroism spectroscopy.

The interaction of mitoxantrone with d-(CTCGAG)2 has been studied by absorption, fluorescence and circular dichroism (CD) spectroscopy. The hypochromism and quenching of fluorescence showed that about four mitoxantrone molecules may be binding externally to DNA hexamer sequence at high drug to nucleic (D/N) acid duplex ratios (28.0-1.1). At lower D/N ratios (1.0-0.2), a red shift in absorption maxima at 610 and 660 nm by 15 and 20 nm, respectively and a red shift in emission maxima by 11 nm accompanied by increase in absorbance and emission has been observed. The equilibrium constant for binding at low (1.0-0.2) and high (28.0-1.1) D/N ratios is 1.8×10(5) M(-1) and 1.38×10(6) M(-1), respectively. The CD spectra show change in intensity of bands accompanied by appearance of induced bands at 325 nm and 650-700 nm. The 251 nm band shows blue shift at D/N ratio of 0.25 and 0.5. The binding isotherms show stoichiometry of 0.25 and 0.5 mitoxantrone molecules binding per duplex. The results suggest stacking of aromatic chromophore of mitoxantrone with terminal base pair of DNA strand forming a sandwiched structure of mitoxantrone between four and two duplex molecules. These investigations are relevant to the formation of ternary complex with topoisomerase enzyme and hence an understanding of anti tumor action of mitoxantrone.

NMR-based structure of anticancer drug mitoxantrone stacked with terminal base pair of DNA hexamer sequence d-(ATCGAT)2.

Mitoxantrone is a promising antitumor drug having considerably reduced cardiotoxicity as compared to anthracyclines. Its binding to deoxyhexanucleotides sequence d-(ATCGAT)2 has been studied by proton and phosphorous-31 nuclear magnetic resonance spectroscopy. The stoichiometry reveals that 1:1 and 2:1 mitoxantrone-d(ATCGAT)2 complexes are formed in solution. Significant upfield shifts in 6H/7H, 2H/3H, 11NH, and 12NH protons (∼.5 ppm) of mitoxantrone and T6NH imino protons (∼.3 ppm) are observed. The phosphorous resonances do not shift significantly indicating that the base pairs do not open at any nucleotide step along the sequence of hexamer. Several inter-molecular Nuclear Overhauser Enhancement connectivities between mitoxantrone and hexanucleotide protons indicate that mitoxantrone chromophore stacks with terminal A1-T6 base pair and side chains involving 12CH2, 12NH, and 14OH protons are in close proximity of A1, T2, A5, and T6 bases. Absorption and emission spectra show red shift in wavelength maxima, which is characteristic of stacking interaction. At higher mitoxantrone to nucleic acid ratios, electrostatic interactions are dominant. The 2:1 drug/DNA stoichiometric structure obtained by restrained Molecular Dynamics simulations shows considerable distortions in backbone torsional angles and helicoidal parameters although structural fluctuations in 25 ps analysis of trajectory are found to be negligible. Mitoxantrone binds as a monomer at either or both ends of hexamer externally with side chains interacting specifically with DNA. The findings are relevant to the understanding of pharmacological action of drug.

A 500 MHz proton NMR study of the conformation of adriamycin

Abstract The conformation of adriamycin has been investigated by proton NMR spectroscopy at 500 MHz in D2O and DMSO. Two-dimensional phase-sensitive COSY spectra are recorded to obtain three-bond and long-range coupling constants and hence torsional angles. Interproton distances are estimated from 2D phase-sensitive NOESY spectra using r(1H–2H) = 2.36 A as internal reference. Pairs of protons correlated by spin-spin coupling give NOE cross peaks with interproton distances

Hexavalent Chromium Effects on Hematological Indices in Rats

Biological interest in chromium stems from its prominent role in industrial pollution and its toxicity to plants and animals. Although chromium, in traces, is known to be essential for the growth and well being of men and animals intakes at higher level have been found to be toxic, mainly to the liver and kidney of experimental animals. Exposure of humans to chromium (vi) is known to cause renal necrosis, hepatic damage and respiratory cancer. In mammals, chromium (vi) caused more damage in liver, kidney and myocardium than did chromium (iii) after an i.p. administration of potassium dichromate or chromium (iii) nitrate. However, the information on its toxicity still warrants further study. The present study reports on the effects of chromium (vi) on hematological indices in rats.

Multispectroscopic methods reveal different modes of interaction of anti cancer drug mitoxantrone with Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT)

The interaction of mitoxantrone with alternating Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT) duplex has been studied by absorption, fluorescence and Circular Dichroism (CD) spectroscopy at Drug to Phosphate base pair ratios D/P=20.0-0.04. Binding to GC polymer occurs in two distinct modes: partial stacking characterized by red shifts of 18-23nm at D/P=0.2-0.8 and external binding at D/P=1.0-20.0 whereas that to AT polymer occurs externally in the entire range of D/P. The binding constant and number of binding sites is 3.7×10(5)M(-1), 0.3 and 1.3× 10(4)M(-1), 1.5 in GC and AT polymers, respectively at low D/P ratios. CD binding isotherms show breakpoints at D/P=0.1, 0.5 and 0.25, 0.5 in GC and AT polymers, respectively. The intrinsic CD bands indicate that the distortions in GC polymer are significantly higher than that in AT polymer. Docking studies show partial insertion of mitoxantrone rings between to GC base pairs in alternating GC polymer. Side chains of mitoxantrone interact specifically with base pairs and DNA backbone. The studies are relevant to the understanding of suppression or inhibition of DNA cleavage on formation of ternary complex with topoisomerase-II enzyme and hence the anti cancer action.

Structure of DNA Hexamer Sequence d-CGATCG by Two-dimensional Nuclear Magnetic Resonance Spectroscopy and Restrained Molecular Dynamics

Abstract Solution conformation of self-complementary DNA duplex d-CGATCG, containing 5′d- CpG 3′ site for intercalation of anticancer drug, daunomycin and adriamycin, has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Complete resonance assignments of all the protons (except some H5′/H5” protons) have been obtained following standard procedures based on double quantum filtered correlation spectroscopy (dQF COSY) and two-dimensional nuclear Overhauser effect (NOE) spectra. Analysis of sums of coupling constants in one-dimensional NMR spectra, cross peak patterns in dQF COSY spectra and inter proton distances shows that the DNA sequence assumes a conformation close to the B-DNA family. The deoxyribose sugar conformation is in dynamic equilibrium with predominantly S-type conformer and a minor N-type conformer with N↔S equilibrium varying with temperature. At 325 K, the mole fraction of the N-conformer increases for some of the residues by ∼ 9%. Using a total of 10 spin-spin coupling constants and 112 NOE intensities, structural refinement has been carried out using Restrained Molecular Dynamics (rMD) with different starting structures, potential functions and rMD protocols. It is observed that pseudorotation phase angle of deoxyribose sugar for A3 and T4 residues is ∼ 180° and ∼ 120°, respectively while all other residues are close to C2′endo-conformation. A large propeller twist (∼ −18°) and smallest twist angle (∼ 31°) at A3pT4 step, in the middle of the sequence, a wider (12 Å) and shallower (3.0 Å) major groove with glycosidic bond rotation as high anti at both the ends of hexanucleotide are observed. The structure shows base-sequence dependent variations and hence strong local structural heterogeneity, which may have implications in ligand binding.

Molecular recognition of parallel DNA quadruplex d‐(TTAGGGT)4 by mitoxantrone: binding with 1:2 stoichiometry leading to thermal stabilization and telomerase inhibition

The interaction of the anthraquinone derivative mitoxantrone, a semisynthetic anti‐cancer drug with two non‐planar side chains, with heptamer G‐quadruplex d(TTAGGGT)4, which contains the human telomere DNA sequence, was evaluated by differential scanning calorimetry, fluorescence Job plotting, absorption, and NMR and CD spectroscopy. Binding led to thermal stabilization of DNA (ΔTm=13–20 °C). The spectra revealed that two mitoxantrone molecules bind externally at two sites of the DNA quadruplex as monomers, by partial insertion of the chromophore and side‐chain interaction at the grooves. The inhibition of telomerase (IC50=2 μm), as determined by a TRAP assay, can be attributed to thermal stabilization of the DNA quadruplex because of the interactions with mitoxantrone. The studies revealed highly specific molecular recognition between a ligand and a parallel‐stranded G‐quadruplex; this might serve as a platform for the rational design of new drugs.

Binding of the alkaloid coralyne to parallel G-quadruplex DNA [d(TTGGGGT)]4 studied by multi-spectroscopic techniques

Binding of coralyne to tetra-molecular parallel G-quadruplex DNA [d(TTGGGGT)]4 was evaluated for the first time using biophysical techniques. Absorbance titrations show hypo/hyper-chromism accompanied by 12nm red shift with binding constant Kb=0.2-4.0×106M-1. Binding induces a negative circular dichroism band of coralyne at 315nm. Quenching of fluorescence (~64%) along with 10nm blue shift in emission maxima indicates proximity of coralyne to guanine bases. Job plot indicates existence of multiple complexes. The observed two fluorescence life times, 6 and 12ns, with relative abundance 33% and 63%, respectively suggest two binding sites/conformations in complex. 1D 1H NMR spectra reveal significant broadening and upfield shift of G3NH, G6NH and G3H8 proton signals upon binding. The NOESY spectra reveal sequence specific non-intercalative interaction of coralyne in monomeric form at two sites close to A3/G3 and G6 bases of [d(TTGGGGT)]4 and [d(TTAGGGT)]4, which has implications in anti-cancer drug action.

Nuclear magnetic resonance studies reveal stabilization of parallel G-quadruplex DNA [d(T2G4T)]4 upon binding to protoberberine alkaloid coralyne

Stabilization of G-quadruplex DNA structures in human telomeric and proto-oncogenic promoter regions upon ligand binding has evolved as a viable anti-cancer strategy. We have studied interaction of coralyne, a human telomerase inhibiting protoberberine alkaloid, with parallel stranded tetrameric G-quadruplex DNA [d(T2G4T)]4 using Circular Dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Appearance of induced CD band and the Diffusion Ordered NMR Spectroscopy (DOSY) experiments confirm the formation of well defined coralyne-DNA complex. 1H and 31P NMR studies reveal that coralyne specifically recognizes T2pG3 and G6pT7 steps in DNA. Guanine imino protons indicate that coralyne binding induces thermal stabilization of the G-quadruplex DNA by >20°C. The observed specific changes and thermal stabilization of DNA upon binding may be attributed to inhibition of telomerase by coralyne.

NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)]4.

The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Jobs method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.