Adriamycin and daunorubicin bind in a cooperative manner to deoxyribonucleic acid

Phase partition techniques have been used to measure the binding of the antitumor drugs adriamycin (NSC-123127) and daunorubicin (NSC-82151) to various DNAs. These methods provide reliable equilibrium binding data at the low levels of drug binding that may be expected in vivo. Both adriamycin and daunorubicin exhibit positive cooperativity (and/or allosterism) in their equilibrium binding to DNA as indicated by the positive slope in the initial region of the binding isotherms (Scatchard plots) under conditions simulating physiological ionic strengths. The cooperative binding (i.e., the appearance of initial positive curvature in the binding isotherms) is dependent upon the ionic strength, which suggests a role for DNA flexibility in the cooperative binding process. An analysis of the slope of the initial portion of the binding isotherms for the interaction of adriamycin with synthetic deoxypolynucleotides shows that the degree of cooperative binding decreases in the order poly(dGdT) X poly(dAdC) greater than or equal to poly(dAdT) X poly(dAdT) greater than poly(dGdC) X poly(dGdC). Marky and Breslauer [Marky, L.A., & Breslauer, K. J. (1982) Biopolymers 21, 2185-2194] found that the average base stacking enthalpies of these synthetic poly-nucleotides were in the same order, which also suggests that the properties of the DNA influence the cooperative binding (and/or allosteric effects). Adriamycin binds with a higher degree of cooperativity than daunorubicin (0.1 M NaCl); although this correlates with the effectiveness of the drugs as antitumor agents, the exact relationship between the observation of cooperative binding and pharmacological activity is yet to be determined.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

On the binding of actinomycin and of daunomycin to DNA: a calorimetric and spectroscopic investigation

The “strong” binding of two antibiotics, actinomycin D and daunomycin, to native DNA (calf-thymus) in dilute aqueous solution has been studied by means of calorimetric and spectroscopic measurements. In essence our results show: (1) Daunomycin interaction with DNA is an exothermic process, all features of which depend in a discontinuous way on the fraction of DNA binding sites engaged by the drug. Fluorescence data indicate that such a discontinuous trend should be independent of the GC content of DNA. (2) Actinomycin binding to DNA is, on the contrary, characterized by a positive enthalpy. For such binding, no discontinuity appears discernible with increasing the molar ratio of drug to DNA (phosphorous) on the basis of calorimetric and fluorescence data. (3) Both antibiotics can be bound simultaneously to DNA: our results would suggest that their binding sites on the biopolymer are independent.Discussion is focussed on the possible information derivable from our data on whether or not intercalation may indeed be the main process through which each antibiotic considered “strongly” interacts with DNA.     

Thermodynamics of the daunomycin-DNA interaction: ionic strength dependence of the enthalpy and entropy

Fluorescence and absorbance methods were used to study the interaction of daunomycin with calf-thymus DNA over a wide range of temperatures and NaCl concentrations. van't Hoff analysis provided estimates for the enthalpy of the binding reaction over the NaCl range of 0.05–1.0 M. Daunomycin binding is exothermic over this entire range, and the favorable binding free energy arises primarily from the large, negative enthalpy. Both the enthalpy change and entropy change are strong functions of ionic strength. Possible molecular contributions to the enthalpy and entropy are discussed, leading to the tentative conclusion that hydrogen-bonding interactions at the interacalation site are the primary contributors to the observed thermodynamic parameters. The dependence of the enthalpy on the ionic strength is well beyond the predictions of current polyelectrolyte theory and cannot be fully accounted for. The enthalpy and entropy changes observed compensate one another to produce relatively small free-energy changes over the range of solution conditions studied.     

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

The melting behavior of a DNA junction structure: a calorimetric and spectroscopic study

We present an investigation of the helix–coil transition in a stable branched oligomer of DNA, known as an immobile DNA junction. This junction is composed of four 16-mer strands, which yield four double-helical arms, each containing 8 nucleotide pairs. Properties of the individual arms of this complex are modeled by four octameric duplexes. We have performed experiments using calorimetry, uv absorbance, and CD spectroscopy to characterize the melting transitions of the junction and each arm. By comparing our spectroscopic and calorimetric results on the junction and its component arms, we are able to conclude the following: (1) The calorimetric transition enthalpy for the overall junction complex is equal to the sum of the calorimetric transition enthalpies of the four constituent duplex arms. (2) The optical and the calorimetric measurements yield qualitatively similar, but not identical thermodynamic data. (3) The melting temperature of the junction is less dependent on concentration than the melting temperatures of the individual arms. We attribute this observation to the tetrameric nature of the junction. (4) The ratio of the calorimetric transition enthalpy of the junction and its corresponding van't Hoff value is close to unity. (5) The CD spectrum of the junction is equal quantitatively to the sum of the B-like CD spectra of the four constituent duplex arms.     

1H-NMR study of the interaction of daunomycin with B-DNA helices of methylated oligodeoxynucleotides.

The interaction of daunomycin with B-DNA double helices of several methylated deoxynucleotides, d(C-G-m5C-G), d(m5C-G-C-G), d(C-G-m5C-G-C-G) and d(m5C-G-C-G-m5C-G) in solution was investigated by 1H-NMR spectroscopy at 500 MHz. At low temperature (t less than 20 degrees C for the tetramer and t less than 40 degrees C for the hexamers), several daunomycin-DNA complexes were observed in slow exchange with the drug-free DNA duplexes. The presence of daunomycin in a self-complementary double helix cancels the conformational symmetry of the two strands; the proton signals can split into several others owing to the difference between free and intercalated duplexes and to the many possible intercalation sites in a duplex (three for a tetramer, five for an hexamer). A model relating the chemical shifts of splitted proton signals to the various intercalated duplex conformations was given. The results show that one daunomycin molecule is associated with one duplex and that it can enter any intercalation site with equal probability; no side-effects were observed even for very short helices (of a tetramer). In the case of d(C-G-m5C-G) the association constant and the dissociation and association rates of the intercalated complex were evaluated.     

Daunomycin binding to deoxypolynucleotides with alternating sequences: complete thermodynamic profiles of heterogeneous binding sites

Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25 degrees C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be -5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.     

The energetics of HMG box interactions with DNA: thermodynamics of the DNA binding of the HMG box from mouse sox-5

The energetics of the Sox-5 HMG box interaction with DNA duplexes, containing the recognition sequence AACAAT, were studied by fluorescence spectroscopy, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). Fluorescence titration showed that the association constant of this HMG box with the duplexes is of the order 4x10(7) M(-1), increasing somewhat with temperature rise, i.e. the Gibbs energy is -40 kJ mol(-1) at 5 degrees C, decreasing to -48 kJ mol(-1) at 32 degrees C. ITC measurements of the enthalpy of association over this temperature range showed an endothermic effect below 17 degrees C and an exothermic effect above, suggesting a heat capacity change on binding of about -4 kJ K(-1) mol(-1), a value twice larger than expected from structural considerations. A straightforward interpretation of ITC data in heat capacity terms assumes, however, that the heat capacities of all participants in the association reaction do not change over the considered temperature range. Our previous studies showed that over the temperature range of the ITC experiments the HMG box of Sox-5 starts to unfold, absorbing heat and the heat capacities of the DNA duplexes also increase significantly. These heat capacity effects differ from that of the DNA/Sox-5 complex. Correcting the ITC measured binding enthalpies for the heat capacity changes of the components and complex yielded the net enthalpies which exhibit a temperature dependence of about -2 kJ K(-1) mol(-1), in good agreement with that predicted on the basis of dehydration of the protein-DNA interface. Using the derived heat capacity change and the enthalpy and Gibbs energy of association measured at 5 degrees C, the net enthalpy and entropy of association of the fully folded HMG box with the target DNA duplexes was determined over a broad temperature range. These functions were compared with those for other known cases of sequence specific DNA/protein association. It appears that the enthalpy and entropy of association of minor groove binding proteins are more positive than for proteins binding in the major groove. The observed thermodynamic characteristics of protein binding to the A+T-rich minor groove of DNA might result from dehydration of both polar and non-polar groups at the interface and release of counterions. The expected entropy of dehydration was calculated and found to be too large to be compensated by the negative entropy of reduction of translational/rotational freedom. This implies that DNA/HMG box association proceeds with significant decrease of conformational entropy, i.e. reduction in conformational mobility.     

Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

DAUNOMYCIN BINDING TO DEOXYPOLYNUCLEOTIDES WITH ALTERNATING SEQUENCES: COMPLETE THERMODYNAMIC PROFILES OF HETEROGENEOUS BINDING SITES

ABSTRACT

Complete thermodynamic binding profiles for the interaction of the anticancer drug, daunomycin with natural DNA and synthetic deoxypolynucleotides were described. Fluorescence titration method was used to estimate the equilibrium binding constants. Binding isotherms were found to be surprisingly complex in some cases, presumably because there were heterogeneous sites even in simple deoxypolynucleotides of repeating sequence. Some polynucleotides consisting of alternating sequence contain at least two different binding sites for daunomycin. The binding affinity of the primary binding sites of alternating and non-alternating sequences was found to differ by two orders of magnitude. An isothermal microtitration calorimeter was used to directly measure the binding enthalpy at 25°C with a high sensitivity. The binding enthalpy of poly[d(A-T)] was found to be ?5.5 Kcal/mol, which was much lower than any other polynucleotides, while the binding constant of the high affinity sites, was similar. In this report, the complete thermodynamic profiles of daunomycin binding to deoxypolynucleotides were reliably shown for the first time.     

Calorimetric and spectroscopic investigation of drug-DNA interactions. I. The binding of netropsin to poly d(AT)

We report the first calorimetric investigation of netropsin binding to poly d(AT). Temperature-dependent uv absorption, circular dichroism (CD), batch calorimetry, and differential scanning calorimetry (DSC) were used to detect, monitor, and thermodynamically characterize the binding process. The following results have been obtained: 1) Netropsin groove binding is accompanied by a large exothermic enthalpy of 9.2 kcal/mol of drug bound at 25 degrees C. This indicates that a large negative binding enthalpy may be a necessary but not a sufficient criterion for drug intercalation. We suggest that the exothermic binding might be correlated with specific H-bonding interactions. 2) From the difference in DSC transition enthalpies in the presence and absence of netropsin, we calculate a binding enthalpy of -10.7 kcal/mol of netropsin at 88 degrees C. 3) We calculate a positive delta S for netropsin binding to poly d(AT) at 25 degrees C. This positive entropy change may reflect netropsin-induced release of condensed cations and/or bound water. 4) The netropsin-saturated duplex monophasically melts 46 degrees C higher than the free duplex. The unsaturated duplex melts through two thermally-resolved transitions that correspond to netropsin-free and netropsin-bound regions. These two regions interact dynamically with no substantial influence on the thermal stabilities of the separate domains. 5) Netropsin binding decreases the cooperativity of the duplex to single strand transition.     

Thermodynamic comparison of PNA/DNA and DNA/DNA hybridization reactions at ambient temperature

The thermodynamics of 13 hybridization reactions between 10 base DNA sequences of design 5'-ATGCXYATGC-3' with X, Y = A, C, G, T and their complementary PNA and DNA sequences were determined from isothermal titration calorimetry (ITC) measurements at ambient temperature. For the PNA/DNA hybridization reactions, the binding constants range from 1.8 x 10(6)M(-1)for PNA(TT)/DNA to 4.15 x 10(7)M(-1)for PNA(GA)/DNA and the binding enthalpies range from -194 kJ mol(-1)for PNA(CG)/DNA to -77 kJ mol(-1)for PNA(GT)/DNA. For the corresponding DNA/DNA binding reactions, the binding constants range from 2.9 x 10(5)M(-1)for DNA(GT)/DNA to 1.9 x 10(7)M(-1)for DNA(CC)/DNA and the binding enthalpies range from -223 kJ mol(-1)for DNA(CG)/DNA to -124 kJ mol(-1)for DNA(TT)/DNA. Most of the PNA sequences exhibited tighter binding affinities than their corresponding DNA sequences resulting from smaller entropy changes in the PNA/DNA hybridization reactions. van't Hoff enthalpies and extrapolated Delta G values determined from UV melting studies on the duplexes exhibited closer agreement with the ITC binding enthalpies and Delta G values for the DNA/DNA duplexes than for the PNA/DNA duplexes.     

Calorimetric and spectroscopic investigation of drug--DNA interactions: II. Dipyrandium binding to poly d(AT).

We report the first calorimetric investigation of steroid diamine binding to a DNA duplex. Absorption spectroscopy, batch calorimetry, and differential scanning calorimetry (DSC) have been used to detect, monitor, and thermodynamically characterize the binding of the steroid diamine, dipyrandium, to poly d(AT). The following thermodynamic data for the binding in 16 mM Na+ at 25 degrees C have been obtained: delta G degree = -6.5 kcal/mol, delta H degree = +4.2 kcal/mol, and delta S = +36 e.u. We interpret the endothermic binding enthalpy in terms of steroid-induced conformational changes in the duplex (e.g. "kinking"). The large positive entropy is interpreted in terms of binding-induced release of bound water and condensed sodium ions. The salt-dependence of the binding constant is interpreted in terms of dipyrandium site-binding involving only one of the two charged ends of the steroid. The optical and DSC curves for the unsaturated steroid-poly d(AT) complexes exhibit biphasic behavior. A comparison of the van't Hoff and the calorimetric transition enthalpies reveals that steroid binding reduces the cooperativity of the transition.     

Kinetics of the daunomycin--DNA interaction

The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.     

Significant discrepancies between van't Hoff and calorimetric enthalpies. II.

Isothermal calorimetric titration of 18-crown-6 ether with BaCl2 in pure aqueous solution over the temperature range 7-40 degrees C gives precise binding constants and enthalpy changes. Nonlinear least-squares fitting of the binding constants to the integrated van't Hoff equation, including a temperature-independent change in heat capacity, leads to van't Hoff enthalpies that differ significantly from the observed calorimetric enthalpies. This perplexing discrepancy appears at present to be very widely occurring.     

Daunomycin interaction with DNA: Microcalorimetric studies of the thermodynamics and binding mechanism

Nucleic acids are an important target for many therapeutics. Small molecules that bind to nucleic acids are important in many aspects of medicines, particularly in cancer chemotherapy. In recent years, many studies have utilized polynucleic acids with various sequences to demonstrate the binding mechanism of daunomycin, a potent anticancer drug. This study describes that isothermal titration calorimetry is a useful tool for studying the fundamental binding mechanism systemically. The results suggest that the binding free energy is more favorable when the temperature is increased. The binding entropy contributes to this effect. Furthermore, the amine group on daunomycin contributes electrostatic interaction that induces the binding process. In addition, enthalpy-entropy compensation is also exhibited in the daunomycin-DNA binding mechanism. This study used an easy, convenient method of performing a systemic study in a recognition system. The results from this study provide additional information about microscopic mechanisms for molecular design and molecular recognition.     

Selective binding of actinomycin D induces a reversible conformational transition of nucleosomes

Equilibrium and hydrodynamic studies on the complex of actinomycin D with H1-H5 depleted, 175 basepair nucleosomes are reported. By spectral titration the intrinsic affinities of actinomycin D for nucleosomes and for DNA are found strictly comparable. Sedimentation analysis shows that actinomycin can apparently unfold the nucleosome, like ethidium bromide and daunomycin, but it does so at a much lower bound drug to DNA molar ratio (about 1 drug molecule to 45 basepairs). Since about four bound actinomycin molecules are able to induce the reversible conformational transition of a nucleosome, it is suggested that the sites of interaction may correspond to the kinked DNA sites evidenced by Klug and collaborators (Richmond, T.J., Finch, J.T., Rushton, B., Rhodes, D. and Klug, A. (1984) Nature 311, 532-537) in the structure of the nucleosome. A relevance of these findings to the interaction of actinomycin with "active chromatin" is also suggested.