Báo cáo khoa học: Structure–activity relation for synthetic phenoxazone drugs Evidence for a direct correlation between DNA binding and pro-apoptotic activity pdf

Structure–activity relation for synthetic phenoxazone drugs Evidence for a direct correlation between DNA binding and pro-apoptotic activity Alexei N. Veselkov 1 , Vladimir Ya. Maleev 2 , Evgenie N. Glibin 3 , Leonid Karawajew 4 and David B. Davies 5 1 Department of Physics and Chemistry, Sevastopol National Technical University, Crimea, Ukraine; 2 Department of Biophysical and Medical Physics, Kharkov National University, Ukraine; 3 Department of Chemistry, St Petersburg State Technological University, Russia; 4 Department of Haematology, Oncology, and Tumour Immunology, Robert-Ro ¨ ssle Clinic, Charite ´ , Humboldt-University of Berlin, Germany; 5 School of Biological and Chemical Sciences, Birkbeck College, University of London, UK The structure–activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. The thermodynamic parameters of drug– DNA complexation were determined by differential scan- ning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity. Minor modifica- tions in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked struc- ture–activity relation. Keywords: apoptotic activity; differential scanning calori- metry (DSC); drug–DNA binding; phenoxazone drugs; structure–activity relationship. Many anti-tumour drugs are thought to exert their cytotoxic effect through DNA-specific interactions, resulting in geno- toxic stress and consequent induction of programmed cell death (apoptosis) [1–3]. Clinically important drugs belong to structurally different families, reflecting the range of possible anchoring mechanisms and their different activities with nucleic acids [4]. These drugs include intercalators, groove binders, and those binding with a combination of the two mechanisms. The antibiotic actinomycin D consists of a planar phenoxazone chromophore with two identical side chains consisting of pentapeptide lactone rings. It is an example of an aromatic drug with both intercalative and groove-binding mechanisms of complexation with DNA. Although the structural significance of the phenoxazone chromophore is well established, the role of the side chains is still under discussion. One hypothesis suggested [5] that actinomycin D may be characterized as an ionophore- antibiotic, because it shows significant complexation of the side chains with sodium ions but not with potassium ions; this, in turn, suggested that the activity of actinomycin D may only be manifested when the pentapeptide rings form complexes with sodium ions. As crown ethers are well known to exhibit selective binding with metal cations [6], this hypothesis was tested on actinomycin D derivatives with crown-like structures in the side chains [7]. None of the derivatives showed significant activity with human leukemia MOLT-3 cell lines, even though the crown side groups had different specificities for metal cation binding, different lengths of spacers in the side chains, etc. [7]. On the other hand, it was found that the rather simple dimethyl- aminoalkylamidophenoxazone derivative (n ¼ 3, Fig. 1) chosen as a standard was reasonably active at the 1 l M level [7]. Interestingly, development of the aminoalkylanthra- quinone family of anti-tumour drugs resulted in a novel synthetic drug, mitoxantrone, with improved characteristics (less cardiac toxicity) compared with natural anthracycline antibiotics such as doxorubicin and daunomycin [8,9]. The role of alkylamino side groups in a number of fluorenone derivatives has also been investigated in terms of the structure–antiviral activity of these drugs [10–12]. This work focuses on the role of aminoalkyl side chains in the biological activity and drug–DNA complexation pro- perties of a series of synthetic phenoxazone compounds with aminoalkyl side chains of different length and with different terminal functional groups. The biological activity of each drug was investigated in terms of induction of apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. This cell line shows wild-type status of the tumour suppressor gene p53 [13]. Given the well-known role of the p53 protein as a key sensor of DNA damage, this cell line is appropriate for investigating the biological effects of drugs with specific binding to DNA. It was found that the series of synthetic phenoxazone compounds with dimethylaminoalkylamido side chains provided the necessary conditions for optimum Correspondence to D. B. Davies, School of Biological and Chemical Sciences, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK. Fax: + 44 207 631 6246, Tel.: + 44 207 631 6238, E-mail: davidbry@ndavies.co.uk Abbreviations: DSC, differential scanning calorimetry; FITC, fluorescein isothocyanate; PI, propidium iodide. (Received 3 January 2003, revised 29 July 2003, accepted 5 September 2003) Eur. J. Biochem. 270, 4200–4207 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03817.x biological activity so that meaningful biophysical studies could be undertaken with a view to understanding the basis of the anticancer activity. The thermodynamic parameters of complexation of the drugs with DNA were determined by differential scanning calorimetry (DSC), which is a con- venient and informative method for obtaining direct data on the thermal stability of drug–DNA complexes. Such information is crucial to the rational design of drugs and for determing the molecular basis of hetero association with other aromatic ligands and their competitive binding with DNA [14,15]. The investigations show that minor modifications in the aminoalkyl side chain of synthetic phenoxazone derivatives (e.g. elongation by one methylene group) lead to consider- able changes in both their anti-tumour activity and DNA- binding properties, providing unambiguous evidence of a marked structure–activity relation. Materials and methods Drugs and DNA A series of actinomycin derivatives with dimethyl- aminoalkyl side chains with different numbers of methylene groups (CH 2 ) n , n ¼ 2, 3, 4, and 5 (Fig. 1) were used to investigate the effect of molecular structure on drug-DNA complexation. The phenoxazone derivatives were synthesized as described previously [16,17] and characterized by IR, UV and 1 H NMR spectroscopy [16–18]. All of the derivatives gave similar experimental values for absorption coefficients at k ¼ 400 nm in the range (1.596–1.603) · 10 4 M )1 Æcm )1 . Therefore ligand con- centrations were determined using the molar absorption coefficient e 400 ¼ 1.6 · 10 4 M )1 Æcm )1 at the isosbestic point of the absorption spectrum. The concentrations of the freeze-dried aromatic compounds determined by weighing were the same as those determined spectrophoto- metrically. For cellular experiments a stock solution of each compound was prepared in dimethyl sulfoxide at a concen- tration of 1 m M . Subsequent dilutions of the drug stock solutions were made in RPMI 1640 medium (Biochrom, Berlin, Germany). Calf thymus DNA (molecular mass > 10 7 Da, charac- terized by a nucleotide content of AT/GC ¼ 1.36 and a level of hyperchromicity of 38–39% at k ¼ 260 nm) was a gift from Professor D. Lando (Institute of Bioorganic Chemistry, Minsk, Belarus). Calf thymus DNA from ÔServaÕ was also used. DNA concentrations were determined spectrophotometrically using a molar absorption coefficient e 260 ¼ 6.4 · 10 3 M )1 Æcm )1 [19]. Solutions of DNA and its complexes with drugs were prepared in 0.1 M NaCl with a phosphate/drug ratio of 5.1–5.5. The concentration of DNA in solution was determined spectrophotometrically at k ¼ 270 nm and k ¼ 290 nm after hydrolysis in 6% HClO 4 solution [20] and was equal to 0.04–0.05%. The corres- ponding molar concentration of DNA phosphates was in the range (1.4–1.7) · 10 )3 M . Aqueous salt DNA solu- tions (0.1 M NaCl) were used in the DSC experiments, pH ¼ 6.5. Cell culture and drug treatment The human leukemia MOLT-3 cell line [13] was obtained from the DSM Cell Culture Bank (Braunschweig, Ger- many). Cells were maintained in RPMI 1640 standard medium containing 2 m ML -glutamine and supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL, Paisley, Scotland, UK). All cultures were free of myco- plasma contamination. To assess drug-induced effects, 0.2 · 10 6 cells per well were cultured in 24-well microtiter plates (Nunc, Roskilde, Denmark) in standard medium at 37 °C in a humidified atmosphere of 5% CO 2 in air [13]. Cells were treated with drugs for 20 h. Assessment of drug-induced apoptosis One of the early events of apoptosis is the loss of membrane asymmetry of phospholipids. At this early stage, the plasma membrane stays intact, but phosphatidylserine, normally located in the inner leaflet of the membrane, redistributes and appears in the outer leaflet. Annexins are a family of proteins that bind to phospholipid membranes in the presence of Ca 2+ . Annexin V binds specifically to phos- phatidylserine on apoptic cell surfaces and can be used as a marker of apoptosis. To determine the extent of apoptosis, cells were stained with fluorescein isothocyanate (FITC)-conjugated annexin V and propidium iodide (PI) using the annexin V kit (Immunotech, Marseille, France) as recommended by the manufacturer. Thereafter, samples were analysed by flow cytometry (FACScan; Becton Dickinson, San Jose, CA, USA) for the presence of viable (annexin V-negative and PI-negative), early apoptotic (annexin V-positive, PI-negative), and late apoptotic (annexin V-positive and PI-positive) cells. The extent of apoptosis was quanti- fied as the percentage of annexin V-positive cells [21]. The extent of drug-specific apoptosis (%) was assessed from: ðdrug-induced apoptosis À apoptosis in mediumÞ100 ð100 À apoptosis in mediumÞ ð1Þ where drug-induced apoptosis is the percentage of annexin V-positive cells in the presence of the drugs, and sponta- neous apoptosis in the medium is the percentage of annexin V-positive cells in control samples [22]. Cytotoxic activity has been defined using calculated values of drug concentrations at which 50% of lethality (drug-specific apoptosis) is achieved, LC 50 . Fig. 1. Chemical structures of the phenoxazone derivatives Act, ActII– ActV. Ó FEBS 2003 Structure–activity of synthetic phenoxazone drugs (Eur. J. Biochem. 270) 4201 Assessment of drug-induced cell cycle perturbations A flow cytometric method developed previously [7] was used to discriminate cell cycle distribution in subpopulations of viable and apoptotic cells identified by specific annexin V staining (annexin V/DNA-staining method). Briefly, cell samples were first stained with FITC-conjugated annexin V and consequently fixed by addition of 2 mL ice-cold 70% ethanol for 1 h at 4 °C. After being washed, the cells were resuspended in 0.5 mL NaCl/P i containing 50 lgÆmL )1 PI, pH 7.5. After treatment with 10 lL10mgÆmL )1 RNase (type I-A; Boehringer Mannheim, Mannheim, Germany) for 30 min at room temperature in the dark, the cells were analysed by flow cytometry. Cell cycle analysis was carried out using CELLQUEST (Becton Dickinson) software. A total of 10 000 and 20 000 cells were characterized by flow cytometry for apoptosis and cell cycle distribution analysis, respectively. All tests were performed in triplicate. DSC Direct measurement by DSC of heat effects caused by the melting of DNA and its complexes with drugs results in determinations of such energy parameters of structural transition as enthalpy change DH, entropy change DS,free energy change DG, melting temperature T m and the interval of melting DT. The calorimetry experiments were carried using a differ- ential scanning microcalorimeter (DASM-4, Pushchino, Moscow Region, Russia) over the working range of temperatures 40–130 °C and with a measuring cell volume of 0.455 mL. The constant impulse power in all measure- ments was 25ÆlW. The solution was kept under an excess pressure of 253 kPa (2.5 atm) to avoid boiling up to 130 °C. The heating rate of all solutions was 1 °CÆmin )1 .TheDSC baseline was recorded for the aqueous salt solution over the temperature range studied. The heat effect of melting of pure DNA and ligand–DNA complexes was calculated from the area under the heat absorption curve with a precision of ± 1%. The melting point T m corresponds to the value of temperature at the maximum of the heat absorption curve. The width of the transition interval DT was determined as a half-width (i.e. width at half height) of the heat absorption curve. All values of thermodynamic parameters were calculated for 1 mol base pairs, taking an average molecular mass of a DNA base pair as 660 Da. Results Dose-dependent apoptosis and cell cycle in the drug-treated leukemia cells The biological activity of the series of phenoxazone deriva- tives Act–ActV (Fig. 1) was assessed by the annexin/ PI method [7]. Fig. 2 shows that the dose-dependent induction of apoptosis depends on the length of the dimethylaminoalkyl side chain. Although all the phenoxa- zone derivatives induce apoptosis at very high concentrations (100 l M ), only ActII (containing two methylene groups, n ¼ 2, in the side chain) and ActIII (n ¼ 3) are significantly effective at lower concentrations (10 l M ), and only ActII is effective at the lowest concentrations tested (£ 1 l M ;Fig. 2). The same systems of drug-treated cells were examined for cell cycle distributions by the annexin/DNA method [7]. Figure 3 shows that the apoptotic effects of the biologically active compounds Act–ActV are associated with cell cycle perturbations, in which similar cell cycle changes, charac- terized by accumulation of cells preferentially in early S-phase and in G2/M-phase, are shown by compounds II, III and IV. However, the concentrations at which the drugs are able to induce cell cycle perturbations depend strongly on the length of the side chain, with ActII being effective at the lowest concentration (1 l M ) whereas ActIII and ActIV are only effective after a 10-fold or 100-fold increase in concentration, respectively. To understand further the molecular basis of the structure–activity relation of this series of phenoxazone drugs, the anti-tumour properties of derivatives with different variations (e.g. amino and diethylamino) in the terminal groups of the aminoalkyl side chains were inves- tigated (compounds 1–7, Table 1). DSC study of thermostability of drug–DNA complexes The results of microcalorimetric measurements of the heat absorption curves q(T) for solutions of pure DNA and complexes with actinocin derivatives ActII–ActV are shown in Fig. 4. The area under the curve of heat capacity dependence on temperature, DC p ¼ ƒ(T), and the baseline drawn between the temperatures at the beginning (T 1 ) and the end (T 2 )of the transition, corresponds to the heat change DQ 0 (enthalpy change DH at constant pressure P) induced by the thermal transition of the biopolymers [23]: DQ 0 ¼ DH ¼ Z T 2 T 1 DC p dT ð2Þ The entropy change (DS) is derived by integration of the following equation: DS ¼ Z T 2 T 1 DC p T dT ð3Þ The change in Gibbs free energy (DG) for the melting of DNA and its complexes with ligands may be calculated from the general thermodynamic relation: DG ¼ DH À TDS ð4Þ The thermostabilities of DNA and its complexes with ligands were investigated using the melting curves Q(T), derived from the heat absorption curves DC p (T) using the following relation: HðTÞ¼DQðTÞ=DQ 0 ð5Þ where DQðTÞ¼ R T T 1 DC p ðTÞ dT is the heat effect measu- red calorimetrically in the temperature range from T 1 to the current temperature T. The melting curves Q(T) obtained from the heat absorption curves q(T) using eqn 5 are shown in Fig. 5. The binding of ligands with natural and model nucleic acids results in an increase in T m and DT of complexes 4202 A. N. Veselkov et al.(Eur. J. Biochem. 270) Ó FEBS 2003 compared with free nucleic acids [24,25]. The melting enthalpy, DH melt , of nucleic acid complexes with either groove binding or intercalating ligands is higher than DH melt of pure nucleic acids, whereas the entropy of ligand binding (DS bind ) can have both positive and negative values, which mainly results from changes in the environment of the hydrated structure of the ligand– nucleic acid complex relative to the free nucleic acid [26]. The results of calculations of the heat stability (melting temperatures, T m , and intervals of melting, DT) of DNA and its complexes are presented in Table 2. A quantitative estimate of the binding parameters was obtained by subtracting the values describing the thermal transition of pure DNA from those derived for the drug– DNA complexes [27]: DH bind ¼ DH ) DH 0 , DS bind ¼ DS–DS 0 , DG bind ¼ DG ) DG 0 (the zero index relates to pure DNA). The thermodynamic parameters of the endo- thermic melting of DNA and its complexes with drugs, calculated using eqns 2–4 and the binding parameters, DH bind , DS bind and DG bind , are summarized in Table 2. Differences in interaction of ActII–ActV with DNA can also be estimated using the binding parameters DH bind , Fig. 2. Dose-dependent induction of apoptosis by the drugs Act–ActV in leukemic MOLT-3cells. Cells were incubated in the presence of different concentrations of the drugs for 20 h at 37 °C. After incubation, cells were stained with FITC-conjugated annexin V (FL1-H) and PI (FL3-H) before flow cytometric analysis. The extent of apoptosis (normalized with respect to spontaneous apoptosis in the absence of drug) was determined by flow cytometry as described in Materials and methods. Ó FEBS 2003 Structure–activity of synthetic phenoxazone drugs (Eur. J. Biochem. 270) 4203 DS bind , DG bind per molecule of drug. Spectrophotometric investigation of actinomine–DNA complexes has shown [28] that intercalation and external binding of ligand with DNA, characterized by the parameter r (the number of mol of ligand per mol of base pairs), depend on the ratio of DNA and ligand concentrations in solution, and at phosphate/drug ratio ¼ 5.5, the value of the parameter r is % 0.33. The relation of DH bind , DS bind , DG bind to r gives the changes in enthalpy, entropy and free energy of binding of ActII–ActV to DNA per mol of ligand (Table 3). Discussion Examination of the cytotoxic effects in leukemic cells showed that cytotoxic activity (Figs 2 and 3) was a function of the number (n)ofCH 2 groups in the side chain (Table 1). The results, expressed in LC 50 units, exhibit a pronounced maximum in cytotoxic activity for n ¼ 2 (Fig. 6). Hence, the anti-tumour activity of Act–ActV is found to be very sensitive to minor modifications in the side chain of actinomycin D derivatives, indicating a direct correlation Fig. 3. Flow cytometric analysis of the cell cycle perturbations induced by the drugs Act–ActV in MOLT-3 cells. Cells were incubated in the presence of different concentrations of drugs for 20 h at 37 °C and analysed by the annexin V/DNA method [7]. Cell cycle distributions in subpopulations of viable (dotted lines) and apoptotic cells (solid lines) are presented as histogram overlays. 4204 A. N. Veselkov et al.(Eur. J. Biochem. 270) Ó FEBS 2003 between structure and activity of the drugs. It is of interest that investigations by stopped-flow spectrophotometry of the relations between binding mode to DNA and the anti- tumour activity of mitoxantrone, ametantrone and its derivatives have shown [9] that variations in the structure of the aminoalkyl side chains of ametantrone analogs had little effect on the kinetic stability of the complexes. It can be seen from Table 1 that a reduction in the cytotoxic effect of the synthetic phenoxazone drugs results from the presence of short side chains (compounds 1 and 2) or having diethyl (compounds 3 and 4) or amino (com- pounds 5, 6, and 7) groups at the terminal sites of the alkylamino side chains instead of dimethyl groups. It follows that the presence of terminal dimethyl groups in the alkylamino side chains in the series of phenoxazone Fig. 4. Heat absorption curves q (JÆs -1 ) as a function of temperature (°C) for solutions of pure DNA and its complexes with ActII–ActV (after baseline correction). The value of calibrating impulse (10 )5 JÆs )1 )is shown for the case of pure DNA, as an example. Fig. 5. Melting curves of calf thymus DNA and its complexes with ActII–ActV in 0.1 M NaCl at pH 6–6.5. DNA concentration is 0.04– 0.05%; DNA phosphate/drug (P/D), 5.1–5.5. Table 1. Anticancer activity (% drug-specific apoptosis in human leukemia MOLT-3 cell lines) of symmetrically substituted synthetic phenoxazone derivatives. Compound R % Apoptosis 1 l M 10 l M 100 l M Act –NH–N(Me) 2 07 51 ActII –NH–(CH 2 ) 2 –N(Me) 2 93 98 100 ActIII –NH–(CH 2 ) 3 –N(Me) 2 485 99 ActIV –NH–(CH 2 ) 4 –N(Me) 2 24 86 ActV –NH–(CH 2 ) 5 –N(Me) 2 33 48 1 –N(Me) 2 12 12 2 –NHCH 3 12 7 3 –NH–(CH 2 ) 2 –N(Et) 2 3 39 100 4 –NH–(CH 2 ) 3 –N(Et) 2 2 75 100 5 –NH–(CH 2 ) 2 –NH 2 15 89 6 –NH–(CH 2 ) 3 –NH 2 12 46 7 –NH–(CH 2 ) 5 –NH 2 32 48 Table 2. Thermodynamic data of helix to coil transition of calf thymus DNA and its complexes with ActII/ActV determined from DSC meas- urements. All thermodynamic parameters are calculated per mol of DNA base pairs. DH and DS,aswellasDH bind and DS bind values were determined at T ¼ T m . Temperatures are given in °C, and changes in enthalpy as kcalÆmol )1 , entropy as calÆmol )1 ÆK )1 , and free energy as kcalÆmol )1 . Sample Helix–coil transition Drug–DNA complexation T m DT DH DS DG 293 –DH bind –DS bind –DG bind 293 DNA 83.3 10.5 7.50 20.8 1.40 – – – ActII–DNA 105.0 21.0 12.3 32.9 2.66 4.8 12.1 1.26 ActIII–DNA 101.3 14.5 10.4 27.7 2.28 2.9 6.9 0.88 ActIV–DNA 99.0 12.0 10.0 27.0 2.10 2.5 6.2 0.70 ActV–DNA 97.3 11.5 9.7 26.1 2.05 2.2 5.3 0.65 Table 3. Binding parameters for ActII/ActV drug–DNA complexation, calculated per mol of ligand at r = 0.33 (ratio of moles of bound ligand to moles of base pairs). Values are mean ± average deviation. Sample –DH bind (kcalÆmol )1 ) –DS bind (calÆmol )1 ÆK )1 ) – DG bind (kcalÆmol )1 ) ActII–DNA 14.5 ± 1.5 37 ± 2 3.8 ± 1 ActIII–DNA 8.8 ± 1.5 21 ± 2 2.7 ± 1 ActIV–DNA 7.6 ± 1.5 19 ± 2 2.1 ± 1 ActV–DNA 6.7 ± 1.5 16 ± 2 2.0 ± 1 Ó FEBS 2003 Structure–activity of synthetic phenoxazone drugs (Eur. J. Biochem. 270) 4205 derivatives is an important factor in their anti-tumour activity. The thermal studies of drug–DNA complexation also show that different lengths of the aminoalkyl side chains in the series of Act–ActV phenoxazone drugs results in different stabilizing effects on the structure of DNA. It can be seen from Table 2 that the stability of all the drug– DNA complexes is higher than that of pure DNA. For example, as shown in Fig. 7, both the melting temperature T m and free energy changes due to melting of the complexes, DG bind , increase nonlinearly with a decrease in the number of methylene groups in the side chains of the drugs, reaching maximum at n ¼ 2. Thus, the DNA-binding affinity for ActII (which contains two CH 2 groups in the side chain and has maximum biological activity, Fig. 6) is much higher than that of ActIII–ActV (containing more than two CH 2 groups in the side chain), indicating that the degree of drug– DNA complexation and the activity of the drug are related processes. NMR studies of the self-association of ActII–ActV have also shown different behavior for ActII compared with the other phenoxazone drugs [18]; namely, the entropy change during self-association of ActII was appreciably smaller than that of ActIII–ActV, which have longer dimethyl- aminoalkyl side chains. This effect is probably due to the differences in electrostatic and hydrophobic interactions in the ActII molecule with short side chains (n ¼ 2) compared with ActIII–ActV, which have longer dimethylaminoalkyl side chains (n > 2) but the same charge. Although there are small, systematic changes in the binding parameters of ActIII–ActV with DNA, it is seen that their characteristic energies of complexation are quite similar (in comparison with ActII), and the average binding free energy change DG bind is % 0.74 kcal per mol base pairs (Table 2) or 2.25 kcal per mol ligand (Table 3). It appears that the binding enthalpy, DH bind , is mainly responsible for the intercalation type of molecular complexation, whereas hydrogen bonds (as a result of direct contact between the chromophore and GC base pairs) and water bridges may also make a significant contribution. The values of the melting entropy, DS, of the complexes are larger than those for pure DNA (Table 2), which is probably due to the more ordered structure of the hydration environment of drug– DNA complexes compared with pure DNA. The effect for ActII–DNA complexation is significantly greater than for complexation of DNA with ActIII–ActV. Table 3 shows that DH bind for ActII–DNA complexa- tion per mole of ligand, 14.5 kcalÆmol )1 ,islargerby % 7kcalÆmol )1 than the mean value for DH bind for complex formation for ActIII–ActV with DNA. Assuming that the nature of intercalation with DNA is similar for all the drugs investigated, then the additional enthalpy of com- plexation found for ActII–DNA may be due to other types of interactions in this system, e.g. the direct contact between cationic groups of the drug and the sugar– phosphate backbone of DNA. This is currently being investigated. In summary, both the biological activity of synthetic phenoxazone derivatives and the thermodynamic properties of drug–DNA complexation revealed a direct and quite marked structure–activity relation, in which significant changes occur with variation of only one methylene group in the dimethylaminoalkyl side chains. Synthetic phenoxa- zone drugs provide an important series of molecules for investigating structure–activity relations. 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(1995) Thermo- dynamic and spectroscopic characterization of the berenil and pentamidine complexes with the dodecanucleotide d(CGCGA TATCGCG) 2 . Z. Naturforsch. 50c, 263–274. 28. Krivtsova, M.A., Moroshkina, E.B., Glibin, E.N. & Frisman, E.V. (1982) DNA interaction with low molecular ligands of dif- ferent structure. II. Complexes of DNA with actinomine and its analogues. Mol. Biol. 16, 149–155. Ó FEBS 2003 Structure–activity of synthetic phenoxazone drugs (Eur. J. Biochem. 270) 4207 . Structure activity relation for synthetic phenoxazone drugs Evidence for a direct correlation between DNA binding and pro-apoptotic activity Alexei N. Veselkov 1 , Vladimir Ya. Maleev 2 ,. width at half height) of the heat absorption curve. All values of thermodynamic parameters were calculated for 1 mol base pairs, taking an average molecular mass of a DNA base pair as 660 Da. Results Dose-dependent. struc- ture activity relation. Keywords: apoptotic activity; differential scanning calori- metry (DSC); drug DNA binding; phenoxazone drugs; structure activity relationship. Many anti-tumour drugs are