ITC derived binding and thermodynamic profiles for the interaction of harmalol to various polynucleotides^a.

a<p>Average of three determinations in CP buffer of 15 mM [Na⁺], pH 6.8. Values of Δ<i>G^o</i> were determined using the equation Δ<i>G^o</i> = −<i>RT ln K</i>_b and <i>T</i>Δ<i>S^o</i> = Δ<i>H^o</i>−Δ<i>G^o</i>. n denotes the binding site size.</p><p>ITC derived binding and thermodynamic profiles for the interaction of harmalol to various polynucleotides^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108022#nt103" target="_blank">a</a>}.</p

Results of competition dialysis experiment in 15 mM CP buffer, pH 6.8 at 25±0.5°C.

<p>The concentration of harmalol bound to each polynucleotide sample is shown as a bar graph. The data given are average of three independent experiments under identical conditions.</p

Effect of varying concentrations (5, 10, 20, 40 and 55 µM) of Harmalol for 72 hours on the viability (MTT) of different cancer cells (HeLa, MDA-MB-231, A549 and HepG2) resulted in a significant dose-dependent reduction in the viability of the cells.

<p>The data are represented as the means ± SEM of three independent experiments. Significant values are calculated against untreated control cells and analyzed with ANOVA test. (*<i>P</i><0.05 vs. untreated).</p

Temperature dependence of the thermodynamic parameters, <i>T</i>Δ<i>S</i>°, Δ<i>H</i>° and Δ<i>G</i>° for the binding of harmalol to (A) poly(dG-dC).poly(dG-dC) (B) poly(dA-dT).poly(dA-dT).

<p>Values of all the parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108022#pone-0108022-t003" target="_blank">Table 3</a>.</p

Temperature dependent thermodynamic parameters for the binding of harmalol to the hetero polynucleotides.

<p>All the data in this table are derived from ITC experiments conducted in 15 mM CP buffer, pH 6.8 and are average of three determinations. <i>Kb</i> and Δ<i>H</i> o values were determined from ITC profiles fitting to Origin 7 software as described in the text. The values of Δ<i>Go</i> and <i>T</i>Δ<i>So</i> were determined using the equations Δ<i>Go</i> = −RTln<i>K</i>b and <i>T</i>Δ<i>So</i> = Δ<i>Ho</i>−Δ<i>Go</i>. All the ITC profiles were fit to a model of single binding sites.</p><p>Temperature dependent thermodynamic parameters for the binding of harmalol to the hetero polynucleotides.</p

Chemical structure of harmalol in 2 and 3D view.

<p>Chemical structure of harmalol in 2 and 3D view.</p

Sequence Specific Binding of Beta Carboline Alkaloid Harmalol with Deoxyribonucleotides: Binding Heterogeneity, Conformational, Thermodynamic and Cytotoxic Aspects

<div><p>Background</p><p>Base dependent binding of the cytotoxic alkaloid harmalol to four synthetic polynucleotides, poly(dA).poly(dT), poly(dA-dT).poly(dA-dT), poly(dG).poly(dC) and poly(dG-dC).poly(dG-dC) was examined by various photophysical and calorimetric studies, and molecular docking.</p><p>Methodology/Principal Findings</p><p>Binding data obtained from absorbance according to neighbor exclusion model indicated that the binding constant decreased in the order poly(dG-dC).poly(dG-dC)>poly(dA-dT).poly(dA-dT)>poly(dA).poly(dT)>poly(dG).poly(dC). The same trend was shown by the competition dialysis, change in fluorescence steady state intensity, stabilization against thermal denaturation, increase in the specific viscosity and perturbations in circular dichroism spectra. Among the polynucleotides, poly(dA).poly(dT) and poly(dG).poly(dC) showed positive cooperativity where as poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) showed non cooperative binding. Isothermal calorimetric data on the other hand showed enthalpy driven exothermic binding with a hydrophobic contribution to the binding Gibbs energy with poly(dG-dC).poly(dG-dC), and poly(dA-dT).poly(dA-dT) where as harmalol with poly(dA).poly(dT) showed entropy driven endothermic binding and with poly(dG).poly(dC) it was reported to be entropy driven exothermic binding. The study also tested the <i>in vitro</i> chemotherapeutic potential of harmalol in HeLa, MDA-MB-231, A549, and HepG2 cell line by MTT assay.</p><p>Conclusions/Significance</p><p>Studies unequivocally established that harmalol binds strongly with hetero GC polymer by mechanism of intercalation where the alkaloid resists complete overlap to the DNA base pairs inside the intercalation cavity and showed maximum cytotoxicity on HepG2 with IC₅₀ value of 14 µM. The results contribute to the understanding of binding, specificity, energetic, cytotoxicity and docking of harmalol-DNA complexation that will guide synthetic efforts of medicinal chemists for developing better therapeutic agents.</p></div

ITC profile for the binding of 1200 µM of (A) poly(dA-dT).poly(dA-dT), (B) poly(dA).poly(dT), (C) poly(dG-dC).poly(dG-dC) and (D) poly(dG).poly(dC) to harmalol (20 µM) at 25±0.5°C, pH 6.8.

<p>Each heat burst curve (in the bottom part of upper panel) is the result of a 1.5 µL sequential injection of the polynucleotide into harmalol (curves at the bottom). The top part of upper panel show the heat burst for the injection of the polynucleotide into the same buffer as control in each experiment (curves offset for clarity). The lower panel represent the corresponding normalized heat data against the molar ratio (P/D) (E, F, G and H). The data points (•-•) reflect the experimental injection heats while the solid line represents the calculated best fit of the data. The values of the various thermodynamic parameters obtained are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108022#pone-0108022-t002" target="_blank">Table 2</a>.</p

<i>In vitro</i>^a cytotoxicity of harmalol (IC₅₀^b µM).

a<p>Data represent the mean values of three independent determinations.</p>b<p>Cytotoxicity as IC₅₀ for cell line is the concentration of compound which reduced by 50% the optical density of treated cells with respect to untreated cells using MTT assay.</p>c<p>Cell lines include non small cell cervical carcinoma (Hela), breast carcinoma (MDA-MB-231), lung carcinoma (A549) and liver carcinoma (HepG2).</p><p><i>In vitro</i>^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108022#nt105" target="_blank">a</a>} cytotoxicity of harmalol (IC₅₀^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108022#nt106" target="_blank">b</a>} µM).</p

Representative CD spectra resulting from the interaction of harmalol, with the polynucleotides in 15 mM CP buffer of pH 6.8 at 25±0.5°C.

<p>(A) Curves (1–6) denote poly(dA-dT).poly(dA-dT) (40 µM) treated with 0, 5.0, 12.0, 15.0, 25.0 and 35.3 µM of harmalol. (B) Curves (1–6) denote poly(dA).poly(dT) (42 µM,) treated with 0, 2.0, 8.0, 12.0, 25.0 and 40.3 µM of harmalol. (C) Curves (1–7) denote poly(dG-dC).poly(dG-dC) (40 µM,) treated with 0, 2.0, 4.0, 8.0, 15.0, 25.0 and 40.3 µM of harmalol. (D) Curves (1–5) denote poly(dG).poly(dC) (45 µM,) treated with 0, 5.0, 16.0, 34.0 and 45.0 µM of harmalol. The expressed molar ellipticity (<i>θ</i>) in each case is based on the polynucleotides concentration.</p