Molecular Determinants of Site-specific Inhibition of Human DNA Topoisomerase I by Fagaronine and Ethoxidine
2000; Elsevier BV; Volume: 275; Issue: 5 Linguagem: Inglês
10.1074/jbc.275.5.3501
ISSN1083-351X
AutoresFabrice Fleury, Alyona Sukhanova, Anatoli Ianoul, Jérôme Devy, Irina Kudelina, Olivier Duval, Alain J.P. Alix, Jean Claude Jardillier, Igor Nabiev,
Tópico(s)Neutropenia and Cancer Infections
ResumoDNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016–50 μm concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(−1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH2 group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects. DNA topoisomerase (top) I inhibition activity of the natural alkaloid fagaronine (NSC157995) and its new synthetic derivative ethoxidine (12-ethoxy-benzo[c]phenanthridine) has been correlated with their molecular interactions and sequence specificity within the DNA complexes. Flow linear dichroism shows that ethoxidine exhibits the same inhibition of DNA relaxation as fagaronine at the 10-fold lower concentration. The patterns of DNA cleavage by top I show linear enhancement of CPT-dependent sites at the 0.016–50 μm concentrations of fagaronine, whereas ethoxidine suppress both top I-specific and CPT-dependent sites. Suppression of top I-mediated cleavage by ethoxidine is found to be specific for the sites, including strand cut between A and T. Fagaronine and ethoxidine are DNA major groove intercalators. Ethoxidine intercalates DNA in A-T sequences and its 12-ethoxy-moiety (absent in fagaronine) extends into the DNA minor groove. These findings may explain specificity of suppression by ethoxidine of the strong top I cleavage sites with the A(+1), T(−1) immediately adjacent to the strand cut. Fagaronine does not show any sequence specificity of DNA intercalation, but its highly electronegative oxygen of hydroxy group (absent in ethoxidine) is shown to be an acceptor of the hydrogen bond with the NH2 group of G base of DNA. Ability of fagaronine to stabilize top I-mediated ternary complex is proposed to be determined by interaction of its hydroxy group with the guanine at position (+1) of the DNA cleavage site and of quaternary nitrogen interaction with top I. The model proposed provides a guidance for screening new top I-targeted drugs in terms of identification of molecular determinants responsible for their top I inhibition effects. DNA topoisomerase I flow linear dichroism surface-enhanced Raman scattering calf thymus camptothecin phosphate-buffered saline linear dichroism base pair(s) The benzo[c]phenanthridine alkaloid fagaronine (Fig.1), isolated from the roots ofFagara zanthoxyloides Lam. (Rutaceae) (1.Messmer W.M. Tin-Wa M. Fong H.H.S. Bevelle C. Farnsworth N.R. Abraham D.J. Trojavek J. J. Pharm. Sci. 1972; 61: 1858-1859Abstract Full Text PDF PubMed Scopus (147) Google Scholar), exhibits antitumor activity against P388 and L1210 murine leukemias in vivo and toward colon 26 (1.Messmer W.M. Tin-Wa M. Fong H.H.S. Bevelle C. Farnsworth N.R. Abraham D.J. Trojavek J. J. Pharm. Sci. 1972; 61: 1858-1859Abstract Full Text PDF PubMed Scopus (147) Google Scholar, 2.Douros J. Suffness M. Recent Results Cancer Res. 1981; 76: 153-175Crossref PubMed Scopus (37) Google Scholar). It has been shown to induce differentiation in murine erythroid Friend cells, human K562 erythroleukemia and promyelocytic HL60 cells (3.Comoe L. Jeannesson P. Trentesaux C. Desoize B. Jardillier J.C. Leukemia Res. 1987; 11: 445-451Crossref PubMed Scopus (26) Google Scholar, 4.Comoe L. Carpentier Y. Desoize B. Jardillier J.C. Leukemia Res. 1988; 12: 667-672Crossref PubMed Scopus (19) Google Scholar, 5.Barret Y. Sauvaire Y. Phytother. Res. 1992; 6: 59-63Crossref Scopus (23) Google Scholar). Fagaronine was proved to be a DNA intercalator (6.Pezutto J.M. Antosiak S.K. Messmer W.M. Slaytor M.B. Honig G.R. Chem. Biol. Interact. 1983; 43: 323-339Crossref PubMed Scopus (98) Google Scholar), it inhibits DNA and RNA polymerase activities and protein synthesis (7.Torres C.A.C. Baez A. Biochem. Pharmacol. 1986; 35: 679-685Crossref PubMed Scopus (31) Google Scholar, 8.Sethi V.S. Cancer Res. 1976; 36: 2390-2395PubMed Google Scholar). Fagaronine also inhibits reverse transcriptases from different sources (5.Barret Y. Sauvaire Y. Phytother. Res. 1992; 6: 59-63Crossref Scopus (23) Google Scholar, 9.Sethi V.S. Sethi M.L. Biochem. Biophys. Res. Commun. 1975; 63: 1070-1076Crossref PubMed Scopus (49) Google Scholar, 10.Riou G.F. Gutteridge W.E. Biochimie (Paris). 1978; 60: 365-379Crossref PubMed Scopus (22) Google Scholar) and was proposed to act through at least two different mechanisms: inhibition of nucleic acid synthesis due to interaction with DNA and inhibition of the elongation step of protein synthesis (7.Torres C.A.C. Baez A. Biochem. Pharmacol. 1986; 35: 679-685Crossref PubMed Scopus (31) Google Scholar). Further studies revealed that fagaronine is able to stabilize top I1 ternary cleavable complexes at low concentrations and to inhibit both top I and top II ternary complexes at higher concentrations (12.Wang L.K. Johnson R.K. Hecht S.M. Chem. Res. Toxicol. 1993; 6: 813-818Crossref PubMed Scopus (140) Google Scholar, 13.Larsen A.K. Grondard L. Couprie J. Desoize B. Comoe L. Jardillier J.-C. Riou J.F. Biochem. Pharmacol. 1993; 46: 1403-1412Crossref PubMed Scopus (131) Google Scholar). The most potent fagaronine derivative nitidine (Fig. 1), isolated from extract of a climbing shrub Zanthoxyulum nitidum (14.Fang S.D. Wang L.K. Hecht J. J. Org. Chem. 1993; 58: 5025-5027Crossref Scopus (173) Google Scholar), was observed to trap both, top I- and II-cleavable complexes. Nearly 100 naturally occurring alkaloids in this class have been isolated from plants, and many more have been synthesized, but they are generally not markedly better than nitidine and fagaronine (15.Janin Y.L. Croisy A. Riou J.F. Bisagni E. J. Med. Chem. 1993; 36 (and references herein): 3686-3692Crossref PubMed Scopus (108) Google Scholar) and do not exhibit any significant activity against solid tumors. It is worth noting that the only benzo[c]phenanthridine alkaloids found thus far to stabilize the top I-cleavable complexes are those that have previously been shown to have antitumor activity in experimental animal models (12.Wang L.K. Johnson R.K. Hecht S.M. Chem. Res. Toxicol. 1993; 6: 813-818Crossref PubMed Scopus (140) Google Scholar). So, new structural analogues of benzophenanthridines, top I inhibitors with an enlarged spectrum of activity, are highly desirable. In terms of structure-activity relationship, two main points can be emphasized: (i) all the compounds synthesized and studied so far carry the iminium charge on the benzo[c]phenanthridine ring (Fig. 1), which seems to be necessary for their biological action, and (ii) the reactivity of the iminium toward nucleophilic attack has been put forward to explain the anti-leukemia activity of these series (15.Janin Y.L. Croisy A. Riou J.F. Bisagni E. J. Med. Chem. 1993; 36 (and references herein): 3686-3692Crossref PubMed Scopus (108) Google Scholar). Recently, the iminium bond electrophilicity within the benzo[c]phenanthridines was shown to be a factor which requires consideration in ternary complex formation with reverse transcriptase (16.Kerry M.A. Duval O. Waigh R.D. Mackay S.P. J. Pharm. Pharmacol. 1998; 50: 1307-1315Crossref PubMed Scopus (23) Google Scholar). The other molecular determinants playing the key role in the benzo[c]phenanthridines anticancer or enzymes-inhibition activity are not known yet and need to be identified. Recently, a new fagaronine derivative ethoxidine (Fig. 1), has been synthesized by one of us (17.Mackay S.P. Comoe L. Desoize B. Duval O. Jardillier J.C. Waigh R.D. Anti-Cancer Drug Des. 1998; 13: 797-813PubMed Google Scholar), and its activity against human immunodeficiency virus, type 1 reverse transcriptase has been described (16.Kerry M.A. Duval O. Waigh R.D. Mackay S.P. J. Pharm. Pharmacol. 1998; 50: 1307-1315Crossref PubMed Scopus (23) Google Scholar). Our recent Raman, surface-enhanced Raman scattering (SERS), and flow linear dichroism (FLD) comparative studies of ethoxidine and fagaronine DNA complexes showed that the new derivative is an intercalator with its DNA binding mode different from that of fagaronine (18.Ianoul A. Fleury F. Duval O. Waigh R. Jardillier J.C. Alix A.J.P. Nabiev I. J. Phys. Chem. 1999; 103: 2008-2013Crossref Scopus (25) Google Scholar). Our preliminary results demonstrate also 10-fold higher top I inhibition activity of ethoxidine, as well as its much lower IC50 values in the human K562 and A549 cancer cell lines, as compared with fagaronine (19.Fleury, F., Sukhanova, A., Devy, J., Ianoul, A., Nabiev, I., Jardillier, J. C., Duval, O., and Baggetto, L. (1999) in Proceedings of AACR Conference "Molecular Determinants of Sensitivity to Antitumor Agents," Vancouver, British Columbia, Canada April 1999, p. 12Google Scholar). In this paper we present the results of the study with a wide range of biochemical and biophysical techniques aiming to: (i) compare mechanisms of top I inhibition by fagaronine and ethoxidine, (ii) correlate sequence specificity and molecular interactions of these compounds within the DNA complexes with their mechanisms of top I inhibition, and (iii) identify molecular determinants of the drug chromophores responsible for their DNA binding and top I poisoning or catalytic inhibition. CT DNA and the double-stranded poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) polymers were purchased from Sigma. Their concentrations in the DNA base pairs were determined by using molar extinction coefficients of 13,200, 13,900, and 13,200 m−1 cm−1, respectively (20.Wells R.D. Larson J.E. Grant R.C. Shortle B.E. Cantor C.R. J. Mol. Bol. 1970; 54: 465-497Crossref PubMed Scopus (500) Google Scholar). CPT was purchased from Sigma and fagaronine was supplied by National Cancer Institute (Bethesda, MD). The synthesis of ethoxidine was described previously (17.Mackay S.P. Comoe L. Desoize B. Duval O. Jardillier J.C. Waigh R.D. Anti-Cancer Drug Des. 1998; 13: 797-813PubMed Google Scholar). Fagaronine and ethoxidine were prepared as 1 mm stock solutions in methanol and were diluted by buffer to desired concentration. DNA and polymers were dissolved in PBS to 5 mg/ml stock solution. Drug-DNA complexes were prepared by mixing the drug stock solutions with the DNA solution in PBS. Plasmid pGEM7Z(f+) and restriction endonucleases were purchased from Promega and Escherichia coli strain "Sure" was purchased from Stratagene. Bovine pancreatic DNase I and Klenow fragment ofE. coli DNA polymerase I were purchased from Sigma and Roche Molecular Biochemicals, respectively. Recombinant 68-kDa human DNA top I was purified to homogeneity from insect cells using a two-step procedure as described (21.Fleury F. Ianoul A. Kryukov E. Sukhanova A. Kudelina I. Wynne-Jones A. Bronstein I.B. Maizieres M. Berjot M. Dodson G.G. Wilkinson A.J. Holden J.A. Feofanov A.V. Alix A.J.P. Jardillier J.C. Nabiev I. Biochemistry. 1998; 37: 14630-14642Crossref PubMed Scopus (18) Google Scholar, 22.Bronstein I. Wynne-Jones A. Sukhanova A. Fleury F. Ianoul A. Holden J. Alix A.J.P. Dodson G. Jardillier J.C. Nabiev I. Wilkinson A.J. Anticancer Res. 1999; 19: 317-328PubMed Google Scholar). Specific activity of top I used in our assays was found to be 1.8 × 106 units/mg, where one unit of activity is an amount of enzyme yielding 100% of relaxation of 300 ng of supercoiled pGEM7Z(f+) plasmid DNA in 30 min at 37 °C. Preparation of the top I DNA substrates in the form of plasmid constructs containing top I-specific and CPT-dependent cleavage sites was described (23.Sukhanova A. Grokhovsky S. Zhuze A. Roper D. Bronstein I. Biochem. Mol. Biol. Int. 1998; 44: 997-1010Google Scholar). The constructs were purified from the cells and analyzed by DNA sequencing method of Sanger (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For 3′-end labeling, plasmid DNA constructs were cleaved withHindIII and ApaI and labeled with [α-33P]dATP in the presence of the Klenow fragment of DNA polymerase I according to (24.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The 3′-labeled DNA fragments were purified by electrophoresis on a nondenaturing 5% (w/v) polyacrylamide gel and isolated by electroelution followed by ethanol precipitation. Cleavage was carried out by incubating 50 units of top I with a 5 μl of the solution of the radioactively labeled DNA fragment (3,000–10,000 cpm) in 10 mm Tris-HCl (pH 7.8), 5% glycerol, 0.5 mmEDTA, 0.3 mm 2-mercaptoethanol (final volume 20 μl). For the analysis of DNA cleavage by top I in the presence of the drugs, reaction mixtures were incubated at 25 °C for 20 min, then SDS and proteinase K were adjusted to 0.5% (w/v) and 1 mg/ml, respectively. After incubation for a further 45 min at 37 °C, DNA was purified by phenol extraction, precipitated with ethanol, washed with 70% ethanol, and dried. The samples of DNA were dissolved in 1.5 μl of the formamide-dye mixture (90% formamide containing 15 mm EDTA (pH 8)), heated 1 min at 90 °C, and applied to 8% denaturing polyacrylamide gel. Electrophoresis was proceeded for 65 min at 65 watts (2,500 V). The gels were fixed with 10% acetic acid and dried on glass pretreated with Bind-silane (Amersham Pharmacia Biotech). Cleavage products were identified by comparison with "A + G" Maxam-Gilbert sequencing ladder. Uv-visible spectra were recorded with a JASCO V-530 UV-visible scanning spectrophotometer. CD spectra were recorded in the region 200–500 nm with a Jobin Yvon Mark III dichrograph. CD and UV-visible measurements were performed using quartz cells of 1 and 0.5 cm, respectively. FLD spectra were recorded in the region 220–450 nm with a Jobin Yvon Mark III dichrograph equipped with a self-made achromatic λ/4 device. The self-made flow cell described in Ref. 21.Fleury F. Ianoul A. Kryukov E. Sukhanova A. Kudelina I. Wynne-Jones A. Bronstein I.B. Maizieres M. Berjot M. Dodson G.G. Wilkinson A.J. Holden J.A. Feofanov A.V. Alix A.J.P. Jardillier J.C. Nabiev I. Biochemistry. 1998; 37: 14630-14642Crossref PubMed Scopus (18) Google Scholar, with optical length ∼0.5 mm and volume 200 μl was used for orientation of DNA in the flow. Inhibition of top I-induced DNA relaxation reaction was monitored using a measurement of the LD signal at 260 nm. 3 μg of pGEM7Zf(+) plasmid in 200 μl of a reaction buffer (10 mm Tris-HCl (pH 7.5), 100 mm NaCl, 0.05 mg/ml bovine serum albumin, 0.5 mm EDTA, 5% glycerol) was placed into the flow cell. Then about 3 units of top I were added directly into the cell. Time kinetics was observed as an increase of the FLD signal. Inhibition of DNA relaxation was performed by addition of fagaronine or ethoxidine stock solutions into reaction mixture to obtain the final concentrations of 4.3 or 0.86 μm fagaronine and 0.45 or 0.22 μm ethoxidine. Linear dichroism (ΔA) is the difference between the absorbance for light polarized parallel (A ∥) and perpendicular (A⊥) to the flow. The reduced linear dichroism (LDr) is defined by LDr = ΔA/A = (A ∥ −A ⊥)/A, where A is the isotropic absorbance of the sample. Measurements of the linear dichroism in the region of absorption of the drug was used to determine the drugs chromophore orientation relative to the DNA axis. The angle β between the transition moment of the dye chromophore and the orientation axis of the DNA was calculated from the measured ratios of LDr for the DNA bases and for the drugs, (ΔA/A) drug/(ΔA/A) DNA=(3cos2β−1)/(3cos2α−1)Equation 1 where α = 86° is the angle between transition moment of the bases and the orientation axis of the DNA molecule (25.Matsuoka Y. Nordén B. Biopolymers. 1982; 21: 2433-2452Crossref PubMed Scopus (74) Google Scholar). SERS spectra were recorded with a spectrometer Coderg, model PHO, with double monochromator in the frequency range 300–1800 cm−1. Ar+-ion laser (Coherent Radiation, model Innova 2020) operating at 457.9 nm (for fagaronine) or at 488 nm (for ethoxidine) wavelengths was used for spectra excitation. SERS spectra were recorded for 1 scan with a 1 s time constant. Silver hydrosol was prepared according to the protocol published before (26.Nabiev I. Chourpa I. Riou J.-F. Nguyen C.H. Lavelle F. Manfait M. Biochemistry. 1994; 33: 9013-9023Crossref PubMed Scopus (70) Google Scholar). The model DNA plasmids with inserted oligonucleotides corresponding to the top I cleavage sites were constructed by modification of the DNA pGEM7Z (f+) plasmid and contain the random combination of the top I recognition sites within the 100–200-bp region (23.Sukhanova A. Grokhovsky S. Zhuze A. Roper D. Bronstein I. Biochem. Mol. Biol. Int. 1998; 44: 997-1010Google Scholar). Restricted DNA fragment of the selected model DNA plasmid 1454 with the strong top I-specific and CPT-dependent cleavage sites (Fig.2) was used as a top I substrate. To compare the abilities of fagaronine and ethoxidine to modulate the top I-mediated DNA cleavage, we have analyzed the DNA cleavage pattern of the α-33P-labeled DNA substrate by enzyme with and without drugs. The cleavable complex formation was estimated by analysis of distribution of the single strand DNA breaks after the SDS and proteinase K treatment. The specific DNA cleavages by top I without any drugs and with 10 μm CPT were used as a control. In the absence of drugs, top I-specific cleavage sites (designated as A, B, C, D, E, G, H) with different cleavage intensities have been revealed (Fig. 2). Addition of 10 μm CPT in the reaction mixture induces typical modulation of the pattern of DNA cleavage by top I. The intensities of cleavage in the sites C and H were found to be unchanged, the sites A, B, D, E, and G were enhanced, and a new site (F) was induced. As expected, all of the enhanced sites have a T base at the cleavage position (−1), and most of them have G at the cleavage position (+1) in accordance with the data published previously (27.Pommier Y. Pourquier P. Fan Y. Strumberg D. Biochim. Biophys. Acta. 1998; 1400: 83-106Crossref PubMed Scopus (550) Google Scholar). Fagaronine induces the same dose-dependent modulation of the top I-mediated DNA cleavage as camptothecin with canonical G(+1) and T(−1) immediately adjacent to the strand cut (Fig. 2). These results confirm the data published previously (12.Wang L.K. Johnson R.K. Hecht S.M. Chem. Res. Toxicol. 1993; 6: 813-818Crossref PubMed Scopus (140) Google Scholar, 13.Larsen A.K. Grondard L. Couprie J. Desoize B. Comoe L. Jardillier J.-C. Riou J.F. Biochem. Pharmacol. 1993; 46: 1403-1412Crossref PubMed Scopus (131) Google Scholar) and were used as a reference to comparative study of the pattern of DNA cleavage modulated by ethoxidine. The pattern of top I-mediated DNA cleavage in the presence of ethoxidine was found to be completely different from that of fagaronine. The presence of ethoxidine in the reaction mixture at the same concentrations as fagaronine induces strong suppression of top I-specific and CPT-dependent cleavage sites (Fig. 2). All these sites were strongly reduced by 2 μm ethoxidine, and some of these sites disappeared at 10 μm, while others at 50 μm concentration of the drug. Suppression of DNA cleavage sites by ethoxidine was found to be sequence-specific. To establish dependence of ethoxidine-induced suppression of DNA cleavage by top I on the local base sequence immediately adjacent to the cleavage site, we have analyzed the site-by-site intensities of DNA cleavage by top I in the presence of ethoxidine. The sites B, C, E, and H include 3′-AT-5′-sequence in position of the strand cut (Group 1), whereas the sites A, D, and G include 3′-GT-5′-sequence in position of the strand cut (Group 2). Densitometric analysis of dependence of DNA cleavage intensities on the drug concentration shows that the sites B, C, E, and, partially, H, are strongly suppressed even at 10 μm ethoxidine concentration (9.1, 15.0, 18.0, and 22.8% of initial cleavage intensity, respectively), whereas the intensities of DNA cleavage in the sites A, D, and G were found to be 33.3, 40.0, and 23.5% from the initial level of DNA cleavage, respectively. An increase of ethoxidine concentration up to 50 μm induces additional preferential suppression of DNA cleavage in the sites B, C, E, H (5.41, 5.0, 16.4, and 16.3% of initial level, respectively), whereas the cleavage in the sites A, D, and G remains significant (27.8, 37.3, and 22.3%, respectively). Obviously, ethoxidine suppresses the sites of the first group more effectively than the sites of the second group. Addition of top I into the flow cell with the plasmid DNA induces enhanced FLD signal saturating within approximately 5 min of DNA/top I incubation (Fig.3 A). In the presence of fagaronine or ethoxidine, kinetics of the DNA relaxation was found to be much slower than in the absence of the drugs. Moreover, at the low drug concentrations 0.22 μm ethoxidine exhibited the same top I inhibitory effect as 0.86 μm fagaronine, whereas at the higher drugs concentrations 0.45 μm ethoxidine exhibited the same effect as 4.3 μm fagaronine (Fig.3 A). Finally, the same level of inhibition of plasmid DNA relaxation by top I was observed at the up to 10-fold lower ethoxidine concentration compared with fagaronine. So, ethoxidine appears to be much more potent top I inhibitor than fagaronine. Fig. 3 B shows reduced linear dichroism as a function of [DNA]/[drug] ratios for fagaronine and ethoxidine DNA complexes. Both curves have the maxima with the highest value of the reduced linear dichroism of fagaronine at 1/2 DNA bp ratio, and of ethoxidine at 1/4 DNA bp ratio. The maxima of the reduced linear dichroism curves indicate the points of DNA "saturation" with the drugs. Thus, fagaronine and ethoxidine interact with the DNA in binding stoichiometry of 1 drug molecule per ∼2.0 bp of DNA and 1 drug molecule per 4.0 bp of DNA, respectively. The FLD technique enables to determine the relative orientation of the plane of the drug chromophore to the plane of DNA bases: the linear dichroism of intercalators is known to be negative, whereas the minor groove binders induce the positive signal (28.Bailly C. Henichart J.P. Colson P. Houssier C. J. Mol. Recognit. 1992; 5: 155-171Crossref PubMed Scopus (77) Google Scholar). FLD signals from fagaronine and ethoxidine bound to DNA at the saturation ratios were found to be negative in the regions of all electronic transitions (Fig.4 B). The angles between the short axis electronic transition of the fagaronine and ethoxidine chromophores and the axis of the DNA molecule calculated with Equation1 are ∼73° and 79°, respectively. So, the plane of the drug chromophores appears nearly normal to the DNA orientation axis, that is almost parallel to the plane of the DNA bases. UV-visible spectra of fagaronine and ethoxidine show two groups of bands corresponding to π → π* electronic transition (Figs. 4 and 5): 1Lb (380–420 nm) and 1La (270–350 nm). The La electronic transition is directed along the long (z axis) of chromophore, whereas Lb lies along its y axis (29.Murrell J.N. The Theory of the Electronic Spectra of Organic Molecules. John Wiley & Sons, New York1963Google Scholar). Deprotonation of fagaronine OH group results in disappearance of bands corresponding to 1Lb electronic transition of the chromophore and an increase with a bathochromic shift of the bands of the 1La transition (Fig. 4 A). The pH dependence of the fagaronine's UV-visible spectrum is determined by its OH group with pK = 8.0 (Fig. 4, inset). Therefore, at the physiological pH the solution contains both protonated and deprotonated forms of fagaronine. UV-visible spectra of ethoxidine in Me2SO, ethanol, methanol, and PBS are found to be practically identical (spectra not shown). On the other hand, the profile and relative intensities of the fagaronine spectra are modified upon Me2SO-ethanol-methanol-PBS transitions (Fig.4 C). The molecules of polar solvents are presumed to form the hydrogen bonds with the oxygen of OH group of fagaronine, and this effect leads to a decrease of the influence of the strong negative charge of the oxygen on the conjugated chromophore system. So, the distribution of electronic density in fagaronine chromophore in polar solutions becomes more similar to this in ethoxidine, and the UV-visible spectra of fagaronine and ethoxidine in PBS are found to be closer than their spectra in Me2SO. The trace 6in Fig. 5 A shows the differential spectra of fagaronine in PBS minus Me2SO. The profile of this difference spectrum may be used as a reference for the effects induced in the case of formation of hydrogen bond between the oxygen of fagaronine's OH group and a less electronegative moiety. Addition of CT DNA to fagaronine or ethoxidine solution results in a hypsochromic shift in their absorption spectra and an increase in the band at ∼400 nm accompanied by relative changes in the bands in the 270–340 nm region (Fig. 5). Characteristic difference spectra of fagaronine and ethoxidine within the DNA complexes are shown in Fig. 5, trace 3. These pronounced spectral modifications induced by DNA binding were used to evaluate the binding constants. For that purpose, DNA was titrated by the drugs (not all the spectral curves are presented in the Fig. 5 for clarity), and the drug/DNA binding constants were determined (Table I).Table IDNA binding constants of fagaronine and ethoxidineDNA/ligandFagaronineEthoxidineCalf thymus DNA1062.5 × 105Poly(dG-dC)·poly(dG-dC)1.1 × 106105Poly(dA-dT)·poly(dA-dT)1062.5 × 106 Open table in a new tab We have analyzed the spectra and determined the binding constants of the drugs within poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) complexes. In terms of the values of its binding constants, fagaronine does not show base preference of intercalation (Table I). The binding constants on the level of 106m−1 were found for its interaction with all used sequences as well as with CT DNA. Contrary, ethoxidine shows strong preference of intercalation within the AT sequences. Specificity of ethoxidine binding with the poly(dG-dC)·poly(dG-dC) is 25-fold lower than with poly(dA-dT)·poly(dA-dT) (Table I). The differences of molecular interactions of fagaronine and ethoxidine within the specific DNA sequences may be revealed from analysis of the profiles of the difference UV-visible spectra of drugs within the complexes (Fig. 5). The spectral profiles of the difference spectra of fagaronine complexes with poly(dG-dC)·poly(dG-dC) and with CT DNA are very similar, and they are drastically different from those of fagaronine-poly(dA-dT)·poly(dA-dT) complexes (compare trace 4 with the traces 3 and 5, Fig.5 A). Otherwise, we did not observe any differences in the spectral profiles of difference spectra of ethoxidine complexed with CT DNA and with a number of alternating polynucleotides (Fig.5 B, traces 3–5). Trace 6 of Fig. 5 A shows the difference spectrum of fagaronine solutions in PBS and in Me2SO. At the same time, the spectra of ethoxidine in Me2SO and PBS solutions were found to be identical (no difference spectrum). Moreover, the profile of the fagaronine difference (PBS minus Me2SO) spectrum in the 1Lb spectral region corresponds exactly to effect of fagaronine binding with poly(dG-dC)·poly(dG-dC) and with CT DNA (compare trace 6 with traces 3 and5 in Fig. 5 A). So, participation of the oxygen of the OH group of fagaronine in the hydrogen bond with the molecules of the polar solvent induces the same spectral effect as its molecular interactions upon DNA and poly(dG-dC)·poly(dG-dC) binding. It is reasonable to suggest that the oxygen of the fagaronine OH group is an acceptor of proton coming from the guanine NH2 group (see "Discussion"). Since the fagaronine and ethoxidine are the planar and achiral chromophores, only those molecules complexed to the asymmetric DNA matrices are able to display induced CD (Fig.6). These induced CD signals, which are indicative of interactions between the drug and host DNA duplex, can be used to detect and to monitor any CD-active DNA binding mode(s). The CD spectra of fagaronine and ethoxidine contain all bands corresponding to 1Lb (400–420 nm) and 1La (270–340 nm) electronic transitions. As is known, the 1La (oriented along chromophore's long axis) and 1Lb electronic transitions lie in the plane of the chromophore's aromatic system and are normal to each other (29.Murrell J.N. The Theory of the Electronic Spectra of Organic Molecules. John Wiley & Sons, New York1963Google Scholar, 30.Schipp
Referência(s)