Thermodynamic characterization of the multivalent binding of chartreusin to DNA

Abstract

Characterization of the thermodynamics of DNA- drug interactions is a very useful part in rational drug design. Isothermal titration calorimetry (ITC), differential scanning calorimetry (DSC) and UV melting experiments have been used to analyze the multivalent (intercalation plus minor groove) binding of the antitumor antibiotic chartreusin to DNA. Using DNA UV melting studies in the presence of the ligand and the binding enthalpy determined by ITC, we determined that the binding constant for the interaction was 3.6 x 10(5) M(-1) at 20 degrees C, in a solution containing 18 mM Na(+). The DNA-drug interaction was enthalpy driven, with a DeltaH(b) of -7.07 kcal/mol at 20 degrees C. Binding enthalpies were determined by ITC in the 20-35 degrees C range and used to calculate a binding-induced change in heat capacity (DeltaCp) of -391 cal/mol K. We have obtained a detailed thermodynamic profile for the interaction of this multivalent drug, which makes possible a dissection of DeltaG(obs) into the component free energy terms. The hydrophobic transfer of the chartreusin chromophore from the solution to the DNA intercalating site is the main contributor to the free energy of binding.

Figure 1

(A) Structural formulae of chartreusin. (B) Continuous variation binding analysis (Job plot) for chartreusin binding to salmon testes DNA. The difference in absorbance at 420 nm as a function of the mole fraction of chartreusin is shown (mean ± SD, three independent experiments). The cross-over point was obtained from linear least squares fits to each data portion, with an inflection point at 0.244 mol fraction, which indicates a 1:3 stoichiometry of drug per DNA (bp).

Figure 2

(A) Representative primary data from an isothermal titration calorimetry experiment, at 30°C, used to obtain multiple estimates of ΔH, in a titration of chartreusin on DNA, using a ‘model-free ITC’ protocol (20) in which a high DNA concentration ensures that all the added ligand is effectively bound. Panels (B)–(E) show the distribution of the DNA-binding enthalpy values for chartreusin, obtained as a function of temperature, at (B) 20°C, (C) 25°C, (D) 30°C and (E) 35°C. The distributions were calculated from three independent ITC analyses at the different temperatures, with 15–25 drug injections in each ITC experiment.

Figure 3

Temperature dependence of the binding enthalpy of chartreusin– DNA interactions. Data are means ± SD of three independent isothermal calorimetric titrations. The slope of the least squares fit of the data renders the heat capacity change, ΔCp, for chartreusin. See Table 1 for further details.

Figure 4

Differential scanning calorimetry curve of salmon testes DNA [excess heat capacity (cal/mol °C) plotted as a function of temperature]. T_m, 74.4 ± 0.1°C; ΔH_wc, 8.8 ± 0.2 kcal/mol. The estimated error is the standard deviation of the mean (three experiments).

Figure 5

(A) Salt dependence of chartreusin binding constants at 20°C. Data are presented according to Record’s theory (22). The linear least squares fit to the data yielded a slope of –0.13. From this value, a negligible positive charge of chartreusin is obtained (Z = 0.15), which is not at variance with the expected uncharged molecule at neutral pH. (B) A tentative parsing of the free energy (ΔG_obs) of chartreusin binding to DNA at 20°C. The ΔG_hyd presented in the figure corresponds to the lower estimate from the equation ΔG_hyd = 80 (± 10) ΔCp (30). Details on all the estimated contributions to the free energy are discussed in the main text.