Interactions of tetracycline and its derivatives with DNA in vitro in presence of metal ions

International Journal of Biological Macromolecules 33 (2003) 49–56

Interactions of tetracycline and its derivatives with DNA in vitro in presence of metal ions Mateen A. Khan a,1 , Javed Musarrat b,∗ b

a Interdisciplinary Biotechnology Unit, A.M.U., Aligarh 20 2002, U.P., India Department of Microbiology, Faculty of Agricultural Sciences, A.M.U., Aligarh 20 2002, U.P., India

Received 9 May 2003; received in revised form 8 July 2003; accepted 8 July 2003

Abstract The interactions of calf thymus DNA with tetracycline (TC), 7-chlorotetracycline (CTC) and 6-dimethyl-7-chlorotetracycline (DMTC) were assessed employing spectrofluorometric and circular dichroism (CD) techniques. The Scatchard analysis revealed relatively lesser binding affinity of TC (Ka = 1.2 × 107 l mol−1 ) vis-a-vis CTC (Ka = 3.4 × 107 l mol−1 ) and DMTC (Ka = 3.0 × 107 l mol−1 ) with DNA. The data suggested both the intercalative and electrostatic nature of binding between the tetracyclines and DNA. The presence of Cu(II) augmented the interaction of tetracyclines with DNA, and resulted in red shift by 12 nm in CD spectra of tetracycline. The molar ellipticity (θ) also changed significantly for CTC and DMTC. The data unequivocally demonstrated the DNA binding potential of tetracyclines both in the presence and absence of Cu(II) ions in dark. The enhanced binding of tetracyclines in presence of Cu(II), ensuing conformational changes in DNA secondary structure to a varying extent, reflects differential reactivity of ligand chromophores. © 2003 Published by Elsevier B.V. Keywords: Tetracyclines; DNA; Binding; Fluorescence; CD spectroscopy

1. Introduction Tetracyclines (TCs) are the broad spectrum antibiotics known to inhibit prokaryotic translation by interfering with binding of the aminoacyl-tRNA to the ribosomal A-site [1,2]. They bind covalently to 30S subunit of ribosomes and polyuridilic acid [3–5], and have also been reported to inhibit the splicing of the Pneumocystis carinii group I intron with a ki of 27 ␮M and group II ␣I5␥ intron of the cox I gene from yeast mitochondria [6]. Furthermore, the self-cleaving activity of the hammerhead ribozyme [7] and the ribozyme derived from the human hepatitis delta virus [8] are also inhibited by tetracyclines. They may also cause alterations in the cytoplasmic membrane, resulting in leakage of nucleotides and other compounds from the cell [9]. Binding of tetracyclines to DNA, RNA and protein in the ∗ Corresponding author. Tel.: +91-571-2502283; fax: +91-571-2703516. E-mail address: [email protected] (J. Musarrat). 1 Present address: Laboratory of Molecular Biology, Department of Chemistry, Hunter College and Graduate Center of the City University of New York, New York 10021, USA.

0141-8130/$ – see front matter © 2003 Published by Elsevier B.V. doi:10.1016/S0141-8130(03)00066-7

presence of divalent cations has also been reported [10,11]. The divalent metal ions play an important role in modulating the biological and biochemical effects of the tetracycline. These effects include the inhibition of certain enzymes [12], the precipitation of ␤-lipoproteins [13] and the localization of tetracyclines in tumour tissues [14]. Earlier studies have demonstrated tetracycline chelation with multivalent cations and their interaction with human red cell membranes, lipids and a variety of proteins using fluorescence and circular dichroism (CD) measurements [15]. Tetracyclines have also been used as fluorescent probe to demonstrate preferential binding to cations on the membrane surfaces [16]. The binding of drugs to DNA has been studied by neighbor exclusion models [17,18], spectroscopic and fluorescence studies [19,20] and DNAse I foot printing [21]. In general, the studies on the binding of various drugs, dyes and antibiotics to DNA have contributed substantially to understanding of their mode of action and site specificity. However, in spite of its prevalent usage, the mode of action of tetracyclines remains poorly understood [22]. Certain studies based on spectral [23,24] and fluorescence [25,26] titration techniques have been used to examine the interactions of ligand chromophores with nucleic acids. These tech-

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niques mainly depend upon the spectral alterations of the ligand upon interaction with nucleic acids [23]. Such alterations have been previously reported for daunomycin [27] and adriamycin [28]. However, to the best of our information, the interaction of tetracyclines with DNA, and the extent and nature of tetracycline-induced alterations in DNA structure have not been extensively studied. Thus, the aim of this study is to address the baseline questions such as: What precisely is the binding affinity of DNA for tetracyclines? How many molecules of tetracyclines specifically interact with DNA nucleotides? and does the metal ions augment the tetracycline-induced structural alterations in DNA? For this purpose sensitive techniques such as fluorescence quenching and CD were used for determining the (i) affinity and stoichiometry of DNA–tetracyclines complexation, (ii) effect of ionic strength and Cu(II) ions on DNA–tetracycline interactions and (iii) extent and nature of induced-conformational changes in DNA upon tetracyclines binding.

increasing concentrations of DNA were added from stock DNA (500 ␮g ml−1 ) solution to obtain the DNA/TCs molar ratios between 0 and 50. The final volume was adjusted to 3 ml with Tris–HCl buffer, pH 7.6. Fluorescence emission spectra were recorded in the wavelength range of 450–570 nm by exciting the TCs at 390 nm. The data were analyzed by Scatchard analysis [30], as described earlier [31] using the following algorithm: nKa − Ka Q =

Q Q = [DNA] [R − Q][Drug]T

(1)

where n is the binding capacity, Ka is the association constant, Q is the fractional quench, [DNA] is the free DNA concentration, [Drug]T is the total drug concentration and R is the DNA/TCs molar ratio. The value of Ka was obtained from the slope of a straight line plot between Q/[DNA] versus Q. 2.4. Effect of ionic strength and Cu(II) ions on DNA–TC interactions

2. Materials and methods 2.1. Materials Calf thymus DNA (sodium salt, average molecular weight one million) was obtained from Sigma Chemical Co., St. Louis, USA. Tetracycline (TC) and its derivatives viz. chlorotetracycline (CTC), oxytetracycline (OTC), doxycycline (DOTC) and demeclocycline (DMTC) were purchased from Hi-Media, India and all other reagents used were of analytical grade. 2.2. Fluorescence spectroscopy

The influence of ionic environment on DNA–TC interaction was assessed at varying NaCl concentrations. Tetracycline (5 ␮M) was allowed to react with increasing concentrations of DNA (0–150 ␮M) in presence of 5, 50 and 200 mM NaCl. Simultaneously, the effect of divalent metal ion, Cu(II) at varying concentrations of 1, 5 and 10 ␮M was also studied. The association constants of tetracycline–DNA complex were determined based on the fluorescence parameters at varying concentrations of monovalent and divalent ions. The number of ion pairs formed between tetracycline and calf thymus DNA (m ), were determined based on the relationship: m ψ =

−∂ logKa ∂ log[M+ ]

(2)

Fluorescence measurements were carried out on a Shimadzu spectrofluorometer, model RF-540 coupled to a data recorder, DR-3 at 25 ± 0.1 ◦ C, using a quartz cell of 1 cm path length. The excitation and emission slits were set at 5 and 10 nm, respectively. The fluorescence of TCs bound to DNA were recorded either in the wavelength range of 450–570 nm or at 525 nm after exciting them at 390 nm. Fluorescence determinations were made for using the fluorescence parameters of Popov et al. [29].

where [M+ ] is the monovalent cation concentration and ψ is the fraction of a counter ion, which is thermodynamically bound per phosphate [32]. Considering [M+ ] equal to [Na+ ], the values for m were determined. The value of ψ as to 0.82, was used to represent the fraction of a counter ion, which is thermodynamically bound per phosphate in a native DNA–drug complex [33].

2.3. Binding data analysis

2.5. Circular dichroism spectroscopy

Binding of TCs to DNA were studied using fluorescence and CD spectroscopy. The TCs binding experiments were performed in 0.01 M Tris–HCl buffer, pH 7.6. All spectral measurements were made after incubating TCs–DNA solutions in dark for 30 min at 25 ◦ C, unless otherwise stated, and the spectra were recorded under subdued light to prevent undesired photodegradation. The fluorescence quenching titration of TCs with increasing DNA/TCs molar ratios were performed in a discontinuous manner. To a fixed concentration (5 ␮M) TCs,

CD spectra of TCs–DNA complex in the presence of Cu(II) ions were measured on a Jasco-720 spectropolarimeter, coupled to a microcomputer. The instrument was calibrated with d-10-camphorsulfonic acid. All the CD measurements were performed using a 1 cm path length quartz cell at 25 ◦ C with a thermostatically controlled cell holder attached to a NESLAB RTE-110 circulating water bath (NESLAB Instruments, Inc., USA) with an accuracy of ± 0.1 ◦ C. All the spectra were collected at a scan speed of 100 nm min−1 with a response time of 1 s. Each sample was scanned three

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times and high frequency noise reduction was applied. Each spectrum was corrected by subtraction of a buffered blank under identical conditions. The results expressed as molar ellipticity (θ)λ in degree cm2 dmol−1 , which is defined as: (θ)λ =

10θ cd

(3)

where θ is the observed ellipticity in degrees, c is the concentration in mol l−1 of total drug, and d is the path length in decimeter.

3. Results and discussion The interactions of TCs with calf thymus DNA were studied by monitoring the changes in the intrinsic fluorescence of TCs at varying DNA/TCs molar ratios. Fig. 1 shows the representative fluorescence emission spectra of TC upon excitation at 390 nm. Addition of calf thymus DNA in increasing concentrations to fixed amount of tetracycline, progressively decreases the fluorescence of TC (Fig. 1). Fig. 2 shows the comparative binding isotherms of tetracycline and its derivatives. The derivatives OTC and DOTC exhibit much less quenching effect upon incubation with DNA compared to other derivatives. It is assumed that

Fig. 1. Fluorescence emission spectra of tetracycline in the absence (uppermost curve) and presence of increasing amounts of DNA after exciting it at 390 nm. The molar ratios of DNA to TC range from 0.0 to 40 (top to bottom) as 0.0, 0.8, 1.6, 3.2, 4.0, 6.0, 8.0, 10, 12, 14, 16, 20, 24, 28, 32, 36, 40.

Fig. 2. Quenching of tetracyclines fluorescence upon DNA binding. Changes in the relative fluorescence of tetracyclines with the addition of DNA are represented as (䊉) TC, (䊊) CTC, (䊏) DMTC, ( ) OTC and (䉱) DOTC.

the amount of fluorescence quenching is proportional to the amount of tetracycline bound to DNA. The Scatchard plots (Fig. 3A–C) provided the association constants as 1.16 × 107 , 3.4 × 107 and 3.04 × 107 l mol−1 , respectively for TC, CTC, and DMTC, respectively. The parameter ‘n’, which is a measure of the number of binding sites of DNA for tetracycline was determined from the intercepts of the straight lines on the X-axis. The variations in the values of n for the tetracycline derivatives account for the differences in their chemical reactivity due to structural variability. Comparative analysis of binding data revealed that CTC and DMTC bind to DNA with three-fold higher affinity than TC. The reduction in intrinsic fluorescence of TCs upon interaction with DNA could be due to masking or burial of tetracycline chromophores upon intercalation between the stacked bases with in the helix and/or surface binding at the reactive nucleophilic sites on the heterocyclic nitrogenous bases of DNA molecule. It is likely that the insertion of planar naphthacene ring of TCs may occur between the adjacent bases of DNA duplex. It could be similar to the intercalation of structurally analogous anthracycline ring as reported in case of anthracyline–DNA interaction [34]. The intercalation of planar molecules between the stacked bases is known to cause alterations in the secondary structure of native DNA duplex. This corroborates well with the observed secondary structural changes in DNA upon tetracycline binding. Our recent studies clearly demonstrated the helix opening and formation of S1 nuclease sensitive sites in tetracycline-modified DNA, which is most likely due to intercalation of tetracyclines [35]. In addition to possible intercalative binding process, the fluorescence data also implicate weaker electrostatic interactions of tetracyclines with DNA.

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Fig. 3. Scatchard plots drawn by fitting the fluorescence quenching titration data for the interaction of DNA with (A) TC, (B) CTC and (C) DMTC. The titration was performed in 0.01 M Tris–HCl buffer, pH 7.6. The drug concentration was 5 ␮M.

Such interactions resulted in the formation of TC–DNA binary complex. However, the extent of binding varies among the tetracycline derivative due to subtle differences in their reactive functional groups. The lesser binding of the parent compound (TC) with DNA vis-a-vis the derivatives CTC and DMTC implies the role of functional groups present on the naphthacene ring system. The derivatives, CTC and DMTC contain a halogen moiety at C-7 position on the D-ring of naphthacene ring system, which might be a contributing factor for their relatively stronger binding affinities. However, the change in number of binding sites of substituted TCs on DNA molecules reflects the relative capacity of tetracycline derivatives to form DNA–TC adducts. To determine the contribution of electrostatic interactions in the overall binding of TCs with DNA, the fluorescence quenching of tetracyclines was measured in solutions of varying ionic strengths. Figs. 4 and 5 show the binding isotherms and Scatchard analysis of TC–DNA interactions in presence of NaCl at varying concentrations. The results indicate that 200 mM NaCl causes about three-fold decrease in the affinity of DNA binding for tetracycline, without significantly altering the binding capacity (Fig. 5). The quenching of tetracycline fluorescence reduces by 27 and 42% at 1:12 DNA–TC molar ratio in presence of 50 and 200 mM NaCl, respectively. Any increase beyond this molar ratio does not alter the quenching effect. The binding constant (Ka ) exhibits a linear relationship with Na+ ion concentration, when log Ka was plotted as a function of −log[Na+ ] (Fig. 6 ). The number of ion pairs formed between TC and DNA (m ) was determined to be 0.675. Another factor that influences the drug–DNA interaction is the relative stability of the DNA duplex. Amongst the forces destabilizing the helical structure of DNA in a solvent, includes the phosphate–phosphate repulsions in the DNA molecule [36]. This effect can be overcome by counter balancing the charge on the phosphate groups of DNA with the Na+ ions. Interestingly, the binding affinity of tetracycline with DNA decreases substantially in the reaction buffers containing NaCl at higher molarities. This could be

Fig. 4. Binding isotherm showing the effect of ionic strength on DNA-induced quenching of tetracycline fluorescence in the presence and absence of NaCl. The curves represent as (䊉) DNA–TC (without NaCl), (䊊) DNA–TC with 5 mM, ( ) DNA-TC with 50 mM and (䉱) DNA–TC with 200 mM NaCl. The excitation and emission wavelength were 390 and 525 nm, respectively.

attributed to poor accessibility of the binding sites on DNA for tetracycline functional groups to bind in presence of Na+ ions. Also the DNA molecule undergoes significant conformational change at higher NaCl concentrations. This may reduce the extent of drug intercalation under higher ionic strength conditions. Earlier studies also demonstrated that at higher concentration of NaCl, the monovalent Na+ ions exhibit a tendency to coordinate with purines, pyrimidines and phosphate groups of DNA, and decreases the unpairing of bases leading to conformational modification of the DNA molecule [36,37]. However, relatively higher binding affinity of tetracycline towards DNA under low ionic strength

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Fig. 5. Scatchard analysis of DNA binding with TC at increasing ionic strength. The plots represent as (A) 0.0 mM, (B) 5 mM, (C) 50 mM and (D) 200 mM NaCl.

Fig. 6. Relationship of the binding constant (Ka ) with ionic strength. The log Ka values obtained for the TC–DNA interactions at varying NaCl concentrations of 5, 50 and 200 mM are plotted as a function of −log[Na+ ].

conditions, implicates the role of electrostatic forces in the DNA–TC interactions. Fig. 7 shows the representative fluorescence emission spectra of tetracycline upon addition of DNA in presence and absence of 10 ␮M Cu(II) ions. Considering the fluorescence intensity value of TC alone as 100%, the extent of quenching at a Cu(II)–TC molar ratio of 2:1 was determined to be 28%. However, the intrinsic fluorescence of TC in combination with DNA alone and DNA–Cu(II) decreased to the extent of 39 and 74%, respectively (Fig. 7). The results clearly exhibit significant DNA–Cu(II) mediated tetracycline fluorescence quenching as compared to DNA or Cu(II) alone. The quantitative data reflecting the quenching effects of tetracycline fluorescence with increasing DNA concentration in the presence of 1, 5 and 10 ␮M Cu(II) ions are shown in Fig. 8. These fluorescence data were analyzed by Scatchard plots (Fig. 9A–D). The value of Ka for TC–DNA complex significantly increases with the addition of Cu(II) ions. This could be possibly due to simultaneous reactions of Cu(II) ions with the oxygen bound at C-3 and amide group as well as the oxygen atoms bound at C-10 and C-11 positions of the TC [38]. Thus, the higher binding affinity of TC–Cu(II) complex with DNA on the intrinsic fluorescence of TC in presence of Cu(II) confirms the role of Cu(II) in promoting the TC complexation with DNA through metal ion bridge formation. However, the

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Fig. 8. Quenching effect of DNA on tetracycline fluorescence in presence of Cu(II) ions. The molar ratio of DNA to TC: 0.0, 0.8, 1.6, 3.2, 4.0, 8.0, 12, 16, 20, 24, 28, 32 and 36. The curves represent as (䊉) control (0.0 ␮M Cu(II)), (䊊) 1 ␮M, ( ) 5 ␮M and (䉱) 10 ␮M.

exhibits a red shift by 10 nm, and a 338 nm (negative) band with a red shift by 12 nm. Addition of DNA to the TC–Cu(II) complex shows slight shifts in (negative) bands. Tetracycline in presence of Cu(II) and DNA separately as well as in combination exhibits significant reduction in ellipticity. The CD spectra of tetracycline, Cu(II) and DNA exhibit an isosbestic point at 383 nm. The CD data of the free tetracyclines and DNA–Cu(II) complex are shown in Table 1. The results indicate a decrease in the molar ellipticity, (θ), for the band maxima and band minima in presence of DNA, to a greater extent for CTC and DMTC as compared to other derivatives. This may be due to the higher binding affinity of these derivatives to DNA. The CD data exhibiting substantial changes in the ellipticity values clearly suggested the structural alterations and formation of TC–Cu(II)–DNA ternary complexes. These ternary complexes may indirectly enhance the surface binding of tetracyclines to DNA due to metal ion bridge formation. The role of metal ion bridges

Fig. 7. Effect of Cu(II) on tetracycline fluorescence. TC fluorescence was measured in absence and presence of 200 ␮M DNA and 10 ␮M Cu(II). The spectra represent as (1) TC alone, (2) TC + Cu(II), (3) TC + DNA and (4) TC + Cu(II) + DNA. Excitation wavelength at 390 nm.

tetracycline–Cu(II) complex upon interaction with DNA transforms into a tetracycline–Cu(II)–DNA ternary complex. The binding data obtained with DNA in the presence of metal ions corroborate well with our earlier studies [31]. The CD spectra of tetracycline in the absence and presence of DNA and Cu(II) ions are shown in Fig. 10. The CD spectra of TC exhibit two characteristic negative CDCEs, both at a shorter and higher wavelengths, and one positive CDCE in between them (curve 1). The results suggest at least two regions of the spectra sensitive to copper ions. These two spectral regions lie in vicinity of 305 nm (positive), which

Table 1 Molar ellipticity (θ) of tetracycline and its derivatives in the presence and absence of DNA and Cu(II) ions Drug

Free drug

Drug complex with DNA and Cu(II)

Band maximum

TC CTC OTC DOTC DMTC

Band minimum

λ (␮m)

(θ) ×

295 297 297 299 299

38.6 42.5 33.4 47.6 39.8

10−5

Band maximum

λ (␮m)

(θ) ×

326 326 327 331 329

15.8 25.6 14.4 16.7 24.9

10−5

Band minimum

λ (␮m)

(θ) ×

305 304 302 313 311

9.7 7.3 9.8 27.8 8.3

10−5

λ (␮m)

(θ) × 10−5

338 340 339 349 349

2.0 9.7 2.1 5.6 10.0

CD spectra were taken in 1 cm path length cells using 50 ␮M of the drug in the presence and absence of DNA (100 ␮M) and Cu(II) (100 ␮M). (θ) is a molar ellipticity and expressed as degree cm2 dmol−1 .

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Fig. 9. Scatchard analysis of TC–DNA binding in the presence of Cu(II) ions. The plots represent as (A) 0.0 ␮M, (B) 1 ␮M, (C) 5 ␮M and (D) 10 ␮M.

Fig. 10. Representative CD spectra of tetracycline (50 ␮M) in 10 mM Tris–HCl buffer, pH 7.4 in the presence and absence of Cu(II) (100 ␮M) and DNA (100 ␮M). The curve (1) TC alone, curve (2) TC + Cu(II) and curve (3) TC + Cu(II) + DNA.

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in enhancing the binding of tetracycline with proteins has been demonstrated [10]. The interactions of the ligands most likely involve the guanine, cytosine and phosphate groups of DNA [39,40]. Thus, in addition to surface binding of tetracycline on DNA, the intercalation of tetracycline may induce substantial conformational changes in the DNA structure.

4. Conclusions The present study demonstrates that the tetracyclines can act both as surface binder and intercalator, involving the electrostatic and hydrophobic binding forces. More hydrophobic tetracyclines such as CTC, DMTC and TC may preferably intercalate into the helix, whereas other derivatives may participate in surface binding on DNA molecule. The data provided important biophysical information related to structure–activity relationship, to help understand the nature of tetracyclines–DNA interactions. It is also suggested that the binding of tetracyclines and Cu(II) with DNA causes perturbations in the secondary structure of DNA, which could be detrimental if the damaged DNA is not accurately repaired. Presumably, the Wilson’s disease patients with genetic disorder of copper metabolism may be predisposed to increased macromolecular damage. Therefore, further studies are warranted to ascertain the extent of tetracycline–DNA–Cu(II) adduct formation, and its influence on the structure and function of certain critical genes implicated in genetic regulation and neoplastic transformations.

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