5.2
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
5.3
Impact Factor
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Search in posts
Search in pages
Filter by Categories
Corrigendum
Current Issue
Editorial
Erratum
Full Length Article
Full lenth article
Letter to Editor
Original Article
Research article
Retraction notice
Review
Review Article
SPECIAL ISSUE: ENVIRONMENTAL CHEMISTRY
View/Download PDF

Translate this page into:

Original article
10 (
2_suppl
); S3947-S3954
doi:
10.1016/j.arabjc.2014.06.001

Interaction of anthelmintic drug (thiabendazole) with DNA: Spectroscopic and molecular modeling studies

,
 Department of Chemistry, Razi University, 67346 Kermanshah, Iran

⁎Corresponding author. Tel./fax: +98 831 4274559. fahimehjalali@yahoo.com (Fahimeh Jalali) fjalali@razi.ac.ir (Fahimeh Jalali)

Received: , Accepted: ,
© The Author(s) 2014
Disclaimer:
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.

Peer review under responsibility of King Saud University.

Abstract

The interaction between thiabendazole (TBZ) and calf-thymus DNA (ct-DNA) was studied by experimental and molecular modeling methods. The intrinsic fluorescence of TBZ was quenched in the presence of ct-DNA. In competition experiments, TBZ could displace Hoechst 33258 (a minor groove binder to DNA), whereas it was unable to replace ethidium bromide (an intercalator). Potassium iodide could quench the fluorescence of TBZ, which indicated the nonintercalative mode of binding of TBZ to ct-DNA. UV absorbance of TBZ shows hyperchromic effect on the addition of DNA to the solution with negligible shift in wavelength. Salt effect studies showed the non-electrostatic nature of binding of TBZ to DNA. The viscosity of ct-DNA solution was almost unchanged on addition of TBZ. Circular dichroism (CD) spectra of DNA showed small changes in the presence of TBZ which is in agreement with groove binding mode of interaction. Moreover, from molecular modeling methods, a docked structure with minimum energy was obtained in which TBZ was located in minor grooves of ct-DNA.

Keywords

Thiabendazole
ct-DNA
Groove binding
Spectroscopy
Molecular modeling
1

1 Introduction

Thiabendazole [2-(4-thiazolyl)benzimidazole (TBZ)], (Scheme 1), is used as a broad spectrum anthelmintic in various animals (Brown et al., 1961) and in humans (Hennekeuser et al., 1969). TBZ inhibits anaerobic respiration at the level of mitochondrial helminth-specific enzyme. This compound has been used also as a food preservative and an agricultural fungicide (Szeto et al., 1993; Arenas and Johnson, 1994). Its multiple uses may cause the entry of its residues into the human food chain, either through direct exposure or through exposure to agricultural products and to food-producing animals. Induction of aneuploidy and photo-genotoxicity has been reported for TBZ in bacteria and cultured human cells (Watanabe-Akanuma et al., 2005).

Chemical structure of thiabendazole (TBZ).
Scheme 1
Chemical structure of thiabendazole (TBZ).

In recent years, the effect of small molecules (drugs, pesticides, fungicides, etc.) on the structure of DNA has attracted the interest of many research groups in the fields of biochemistry and analytical chemistry, because some of them have been categorized as potent genotoxicants and alkylating agents for DNA (Zhu et al., 2014; Zhang et al., 2005, 2013; Kashanian et al., 2008; McCarroll et al., 2002). Some pesticides such as heptachlor, endosulfan, azinphosmethyl and imidacloprid have been reported to interact with DNA and form promutagenic DNA adducts (Laouedj et al., 1995; Shah et al., 1997). Therefore, the studies on the interaction of toxic small molecules with DNA will be helpful in understanding the mechanisms of DNA damage by them and planning the design of more efficient compounds. Intercalation, groove binding, and electrostatic interactions are the three major interaction modes of small molecules with DNA (Haq, 2002; Zeglis et al., 2007). Intercalation occurs when ligands of an appropriate size and chemical nature fit themselves in between base pairs of DNA, while major and minor groove binders interact with the grooves of the double helix of DNA. Electrostatic binding is the interaction between cationic species and the negatively charged DNA phosphate backbone that occurs along the external DNA double helix.

In this work, in vitro interaction between TBZ and DNA was investigated under simulated physiological conditions (Tris–HCl buffer, pH 7.4). UV–Vis absorption, fluorescence and CD spectroscopies, as well as viscosity measurements were used in this study. The binding mode and thermodynamic characteristics of the interaction were discussed using the experimental results and molecular modeling methods.

2

2 Materials and methods

2.1

2.1 Apparatus

Fluorescence measurements were performed with a JASCO spectrofluorimeter Model FP6200 equipped with a Xenon lamp (150 W) and a thermostat bath. UV–Vis absorption spectra were recorded using Agilent 8453 spectrophotometer (Waldbornn, Germany) and a quartz cell with a path length of 1.0 cm. Circular dichroism (CD) measurements were recorded on a JASCO (J-810) spectropolarimeter (Tokyo, Japan). The viscosity measurements were carried out using a SCHOT AVS 450 type viscometer (Germany).

All experiments were run at room temperature (25 ± 0.1 °C), unless specified otherwise.

2.2

2.2 Chemicals

Calf thymus DNA (ct-DNA) was obtained from Sigma and was dissolved in Tris–HCl buffer solution. The purity of DNA was checked by monitoring the ratio of the absorbance at 260 and 280 nm. The solution gave a ratio of A260/A280 > 1.8, which indicates that DNA was sufficiently free from protein (Ling et al., 2008). The concentration of DNA in stock solution was determined by UV absorption at 260 nm using a molar absorption coefficient ε260 = 6600 L mol−1 cm−1 (Kanakis et al., 2009). The prepared DNA solution was stored at 4 °C and used within 4 days. TBZ (Sigma, purity >99%) stock solution (1.0 × 10−2 mol L−1) was prepared by dissolving its pure powder in absolute ethanol solution. Ethidium bromide (EB) (Sigma) stock solution (1.0 × 10−3 mol L−1) was prepared by dissolving in Tris–HCl buffer solution and stored in a cool and dark place. All solutions were adjusted to pH 7.4 with Tris–HCl buffer solution (0.05 mol L−1). Other chemicals were of analytical reagent grade and used without further purification. Doubly distilled deionized water was used throughout.

2.3

2.3 Procedures

2.3.1

2.3.1 Fluorescence measurements

A solution of TBZ (3.0 × 10−5 mol L−1) was titrated by successive additions of a stock solution of DNA. Each solution was allowed to reach equilibrium for 5 min. The fluorescence spectra of the mixtures were then recorded in the wavelength range of 310–530 nm when excited with λex = 295 nm. The emission spectra were recorded at three different temperatures, i.e., 288, 298 and 310 K.

2.3.2

2.3.2 Competition experiments

Increasing amounts of TBZ were injected into a cell containing EB (2.0 × 10−5 mol L−1) and DNA (8.0 × 10−5 mol L−1) at room temperature. After an equilibration interval (2 min), the fluorescence spectra of the mixtures were recorded in the range of 550–670 nm (λex = 530 nm). All fluorescence emission measurements were carried out in 0.05 mol L−1 Tris–HCl buffer (pH 7.4) at room temperature.

In another experiment, TBZ was injected into a cell containing Hoechst 33258 (5.0 × 10−6 mol L−1) and DNA (1.43 × 10−4 mol L−1) at room temperature. Fluorescence spectra of the mixture were recorded in 360–560 nm when excited with λex = 340 nm. The excitation and emission slits were 5 and 10 nm, and the scan speed was 500 nm min−1.

In order to eliminate the inner filter effects of DNA and TBZ, absorbance measurements were performed at excitation and emission wavelengths of fluorescence measurements. The fluorescence intensity was corrected for absorption using the following equation (Zhenxing and Rutao, 2011; Shyamali et al., 1979):

(1)
F cor = F obs 10 A 1 + A 2 / 2 where Fcor and Fobs are the corrected and observed fluorescence, respectively, and A1 and A2 are the sum of the absorbances of DNA and TBZ at excitation and emission wavelengths, respectively.

2.3.3

2.3.3 Iodide quenching experiments

Different concentrations of potassium iodide solution (0.0–2.5 × 10−3 mol L−1) were added to TBZ (3.0 × 10−6 mol L−1) and TBZ–DNA mixture. The fluorescence intensity was recorded after each addition and quenching constants were calculated from the resulted data.

2.3.4

2.3.4 Effect of ionic strength on the fluorescence spectra

A series of assay solutions containing various amounts of sodium chloride (0–0.6 mol L−1) and fixed amounts of TBZ–DNA and TBZ were prepared to study the salt effect by comparing the fluorescence spectra at different ionic strengths.

2.3.5

2.3.5 UV–Vis measurements

UV–Vis absorption spectra of TBZ solution were recorded after successive additions of ct-DNA in the wavelength range of 200–600 nm. All measurements were carried out in Tris–HCl buffer (0.05 mol L−1, pH 7.4) at room temperature (25 ± 0.1 °C).

2.3.6

2.3.6 Circular dichroism (CD) studies

CD spectra of DNA incubated with different amounts of TBZ were measured in the range of 220–320 nm (r = [TBZ]/[DNA] = 0, 1.2, 1.5). The changes in CD spectra were monitored against a blank as ellipticity in mdeg. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before use and kept in a nitrogen atmosphere during experiments. All CD measurements were carried out in Tris–HCl buffer (pH 7.4) at room temperature.

2.3.7

2.3.7 Viscosity measurements

Viscometric titrations were performed using a viscometer, which was immersed in a thermostatic water-bath at 25.0 ± 0.1 °C. The experiments were conducted by adding appropriate amounts of TBZ into the viscometer to give a certain r (=[TBZ]/[DNA]) value while keeping the DNA concentration constant. The flow time of the solution through the capillary was measured with an accuracy of ±0.20 s by using a digital stop watch. The mean value of three replicate measurements was used to evaluate the average relative viscosity of the sample. The data were presented as (η/η0)1/3 versus r (Sun et al., 2008), where η and η0 are the viscosities of DNA in the presence and absence of TBZ, respectively. Viscosity values were calculated from the observed flow time of DNA – containing solutions (t) and corrected for buffer solution (t0), η = (t − t0)/t0.

2.3.8

2.3.8 Molecular docking study

MGL tools 1.5.4 with AutoGrid 4 and AutoDock 4 (Morris et al., 2008, 2009) were used to set up and perform blinded docking calculations between TBZ and a DNA sequence.

DNA sequence d(CGCGAATTCGCG)2 dodecamer (PDB id: 1BNA) was obtained from the Protein Data Bank. Receptor (DNA) and ligand (drug) files were prepared using AutoDock Tools. DNA was enclosed in a box with the number of grid points in x × y × z directions equal to 106 × 100 × 76 and a grid spacing of 0.375 Å. The center of the grid was set to 14.719, 20.979, and 8.824 Å. Lamarckian genetic algorithms, as implemented in AutoDock, were employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy docked conformation, according to the Autodock scoring function, was selected as the binding mode. The output from AutoDock was rendered with PyMol (DeLano, 2004).

3

3 Results and discussion

3.1

3.1 Fluorescence quenching studies

TBZ showed a strong emission band centered at 353 nm when excited with a 295 nm wavelength. The emission spectra of TBZ in the absence and presence of DNA are shown in Fig. 1A. Regular decrease in fluorescence intensity of TBZ is observed with increasing concentration of DNA, which means that DNA quenches the intrinsic fluorescence of TBZ. The quenching rate constant (kq) was calculated according to Stern–Volmer equation:

(2)
F / F 0 = 1 + k q τ 0 [ Q ] = 1 + K SV [ Q ] where F0 and F are the fluorescence intensities in the absence and presence of DNA. KSV is the Stern–Volmer quenching constant, [Q] is the concentration of DNA and τ0 is the average fluorescence lifetime (∼10−8 s).
Fluorescence spectra of TBZ in the presence of increasing amounts of DNA (pH = 7.4, T = 298 K, λex = 295 nm). [TBZ] = 3.0 × 10−5 mol L−1 and [DNA] = 0–1.8 × 10−4 mol L−1.
Figure 1
Fluorescence spectra of TBZ in the presence of increasing amounts of DNA (pH = 7.4, T = 298 K, λex = 295 nm). [TBZ] = 3.0 × 10−5 mol L−1 and [DNA] = 0–1.8 × 10−4 mol L−1.

The plot of Stern–Volmer equation is shown in Fig. 2A, from which kq was obtained to be 4.13 × 1011 M−1 s−1 at 25 °C. For dynamic quenching, the maximum collisional quenching constant obtained for various quenchers is 2.0 × 1010 M−1 s−1 (Wang et al., 2009). However, kq is much larger, suggesting that the quenching is due to the formation of a complex between TBZ and DNA, i.e., static quenching (Sun et al., 2012).

(A) Stern–Volmer plot for quenching of TBZ by DNA (Q) at 25 °C. (B) Plot of log (F0 − F)/F versus log [Q]. (pH = 7.4, λex = 295 nm, λem = 357 nm). [TBZ] = 3.0 × 10−5 mol L−1.
Figure 2
(A) Stern–Volmer plot for quenching of TBZ by DNA (Q) at 25 °C. (B) Plot of log (F0 − F)/F versus log [Q]. (pH = 7.4, λex = 295 nm, λem = 357 nm). [TBZ] = 3.0 × 10−5 mol L−1.

The apparent binding constant (KA) and the binding stoichiometry (n) of DNA–TBZ complex can be estimated by using the following relationship (Li and Dong, 2009):

(3)
log ( F 0 - F ) / F = log K A + n log [ Q ]

The value of KA (t = 25 °C) was obtained from the intercept of the plot of log [(F0 − F)/F] against log [Q] (Fig. 2B) and the stoichiometry (n) from the slope, which were 6.32 × 103 and 1.05, respectively. Because KA is lower than that of classical intercalative binding mode (Shi et al., 2011), groove binding is more reasonable.

3.2

3.2 Study of displacement of Hoechst 33258 by TBZ In DNA

Hoechst 33258, is a synthetic N-methylpiperazine derivative that binds strongly to the minor grooves of DNA with specificity for A–T rich sequences (Murray and Martin, 1988a,b; Haq et al., 1997; Harshman and Dervan, 1985). The quantum yield of Hoechst 33258 increases significantly in the presence of DNA, due to its higher planarity in DNA grooves (compared to the free Hoechst), as well as protection from collisions with solvent molecules (Guan et al., 2006; Weisblum and Haenssler, 1974). Competing ligands for the same sites on DNA, on the other hand, liberate Hoechst to the solvent along with an obvious decrease in the fluorescence intensity of the solution.

Fig. 3 shows the fluorescence spectral changes of bound Hoechst to DNA by addition of TBZ. Comparison with the fluorescence spectrum of TBZ (Fig. 1) shows small overlapping, which seems unimportant due to the different excitation wavelengths of Hoechst and TBZ (340 nm versus 295 nm). The obvious decrease in the fluorescence intensity of bound Hoechst confirms that TBZ is an A–T rich minor groove binder to ct-DNA.

Displacement of Hoechst 33258 (a) in Hoechst–DNA (b) by increasing amounts of TBZ in Tris–HCl buffer (pH 7.4); [Hoechst] = 5 μmol L−1; [DNA] = 1.43 × 10−4 mol L−1; [TBZ] = 2.4–66.7 μmol L−1; λex = 340 nm.
Figure 3
Displacement of Hoechst 33258 (a) in Hoechst–DNA (b) by increasing amounts of TBZ in Tris–HCl buffer (pH 7.4); [Hoechst] = 5 μmol L−1; [DNA] = 1.43 × 10−4 mol L−1; [TBZ] = 2.4–66.7 μmol L−1; λex = 340 nm.

3.3

3.3 Competitive binding of ethidium bromide (EB) and TBZ for ct-DNA

Ethidium bromide (EB) is a well-known intercalator, which is often used as a spectral probe to establish the mode of binding of small molecules to double-helical DNA (Warning, 1965; Lepecq and Paoletti, 1967; Wu et al., 2002). The fluorescence of EB increases after binding to DNA due to intercalation. Like EB, if TBZ intercalates into the helix of DNA, it would compete with EB for the intercalation sites in DNA, and lead to significant decrease in the fluorescence intensity of EB–DNA complex (Ghaderi et al., 2007). For example, lucigenin, as an intercalator, causes 50% decrease in the emission intensity of EB–DNA (Wu et al., 2002). Fig. 4A shows the effect of increasing concentrations of TBZ on the fluorescence intensity of EB bound to ct-DNA. It is apparent from Fig. 4A that, no significant displacement of EB occurs indicating that TBZ did not intercalate with DNA.

(A) Fluorescence intensity of EB–DNA complex in the absence and the presence of TBZ. [DNA] = 4.0 × 10−5 mol L−1; [EB] = 3.2 × 10−6 mol L−1; [TBZ] = 0.0–6.83 × 10−5 mol L−1. (B) Stern–Volmer plots for quenching of TBZ by KI in the absence and presence of DNA. c(TBZ) = 3.0 × 10−6 mol L−1, c(DNA) = 2.0 × 10−4 mol L−1, pH = 7.4.
Figure 4
(A) Fluorescence intensity of EB–DNA complex in the absence and the presence of TBZ. [DNA] = 4.0 × 10−5 mol L−1; [EB] = 3.2 × 10−6 mol L−1; [TBZ] = 0.0–6.83 × 10−5 mol L−1. (B) Stern–Volmer plots for quenching of TBZ by KI in the absence and presence of DNA. c(TBZ) = 3.0 × 10−6 mol L−1, c(DNA) = 2.0 × 10−4 mol L−1, pH = 7.4.

3.4

3.4 Iodide quenching studies

The fluorescence quenching studies give an idea about the availability of the probe molecules to the quencher. In this study, the fluorescence quenching of TBZ was explored using KI as the ionic quencher. Intercalative binding of small molecules to DNA base-pairs shields the molecules from the ionic quenchers, thus showing very low quenching compared to that in aqueous solution (Kumar and Asuncion, 1992). On the contrary, in the case of groove binding, the molecules are exposed to the aqueous solution, thus making the molecule quite available to the quenchers.

In this study, the fluorescence characteristics of TBZ solution were compared with DNA–TBZ mixture during gradual addition of KI. Obviously, KI quenched the emission of TBZ (Fig. 4B) in the absence (Ksv ≈ 87.75) and in the presence of DNA (120.08). These results provide direct evidence (Manna and Chakravorti, 2012) for the groove binding of TBZ with DNA.

3.5

3.5 Thermodynamic parameters and binding forces

The main binding forces between a small molecule and macromolecules in aqueous solution include van der Waals and electrostatic interactions as well as the hydrophobic effect, i.e. the release of water molecules from the solvation shell to the bulk solvent. Timasheff (1972), Ross and Subramanian (1981) characterized the relationship between the sign and magnitude of the changes in thermodynamic parameters (ΔH and ΔS) and the kind of binding forces involved.

In order to calculate ΔH and ΔS, van’t Hoff equation (Eq. (4)) was used. The Gibbs free energy change (ΔG) was determined from the binding constant at a particular temperature according to Eq. (5), and ΔS was estimated according to Eq. (6):

(4)
ln K A 2 / K A 1 = ( 1 / T 1 - 1 / T 2 ) Δ H / R
(5)
Δ G = - RT ln K A
(6)
Δ G = Δ H - T Δ S KA1 and KA2 are the binding constants at temperatures T1 (298 K) and T2 (310 K), respectively.

The calculated negative value of ΔG (−21.68 kJ mol−1) revealed that the interaction between TBZ and DNA is spontaneous in aqueous solution. Positive values of ΔH (26.16 kJ mol−1) and ΔS (160.54 J mol−1 K−1) indicate that the binding process is mainly entropy driven. The fact that ΔH and ΔS are both positive suggests a strong contribution of the hydrophobic effect (Bi et al., 2009).

3.6

3.6 Effect of the ionic strength

DNA is an anionic polyelectrolyte with phosphate groups. Monitoring the spectral change with different ionic strength is an efficient method to distinguish the binding modes between molecules and DNA. NaCl is used to control the ionic strength of the solutions. Due to the competition for phosphate groups, the addition of Na+ would weaken the electrostatic interaction between molecules and DNA [Wu et al., 2009]. The effect of NaCl on the fluorescence of TBZ-DNA system and free TBZ was studied. The results displayed that the fluorescence intensity of TBZ-DNA system and free TBZ did not significantly change with increasing concentration of NaCl (from 0 to 0.6 mol L−1), which suggested non-electrostatic binding of TBZ to DNA.

3.7

3.7 UV absorption spectra of TBZ in the presence of ct-DNA

The absorption spectra of TBZ in the absence and presence of increasing amounts of DNA are given in Fig. 5. Two bands centered at 243 and 297 nm dominated UV region of the electronic spectrum of TBZ. The absorbance of TBZ increased slightly with successive additions of DNA, suggesting that there exists an interaction between TBZ and DNA which is different from the intercalation binding mode. Classical intercalation has been characterized by large changes in the absorbance intensity and wavelength, whereas groove binders always display small changes (Kalanur et al., 2009; Guo et al., 2007). As a consequence, groove binding is suggested as the main mode of interaction of TBZ with ct-DNA.

Absorption spectra of TBZ in the absence and presence of increasing amounts of DNA (1–5), [TBZ] = 5.0 × 10−5 mol L−1, [DNA] = 5.0 × 10−6–7.5 × 10−5 mol L−1. (T = 25 °C). Red spectrum (dashed) is for ct-DNA.
Figure 5
Absorption spectra of TBZ in the absence and presence of increasing amounts of DNA (1–5), [TBZ] = 5.0 × 10−5 mol L−1, [DNA] = 5.0 × 10−6–7.5 × 10−5 mol L−1. (T = 25 °C). Red spectrum (dashed) is for ct-DNA.

3.8

3.8 Circular dichroism (CD) spectroscopy

The morphology changes of DNA can be studied efficiently by CD spectroscopy during its interaction with other species. The CD spectra of free ct-DNA (50 μM) shows a positive band at 273 nm due to base stacking and a negative band at 245 nm due to right handed helicity, this is a characteristic of the B-form of DNA (Novakova et al., 2003; Curtis-Johnson, 1994). These bands are highly sensitive toward the DNA interaction with small molecules (Uma Maheswari and Palaniandavar, 2004; Ivanov et al., 1973). The changes in the CD spectra can be attributed to the corresponding changes in the ct-DNA structure (Lincoln et al., 1997). In the case of minor groove binding, the CD spectra show almost no change, whereas the intercalative binding affects both the positive and negative bands, as observed for classical intercalators, such as methylene blue (Norden and Tjerneld, 1982).

In the present study, the CD spectra of ct-DNA in the absence and presence of increasing concentrations of TBZ were recorded (Fig. 6). Although slight decrement (shifting to zero level) in the negative DNA band is obvious, which indicates decrease in base-pair helicity, however, perturbations on the helicity and especially base-stacking bands are not as pronounced as being indicative of a transition in DNA conformation. This evidence supports groove-binding nature of TBZ-DNA interaction.

Relative specific viscosity of DNA in the presence of TBZ; r = [TBZ]/[DNA].
Figure 6
Relative specific viscosity of DNA in the presence of TBZ; r = [TBZ]/[DNA].

3.9

3.9 Viscosity measurements

Further information on the nature of the binding of TBZ to DNA can be obtained through hydrodynamic studies such as viscosity measurements. Classical intercalation results in lengthening of DNA due to the separation of base-pairs at the intercalation site, which produces a concomitant increase in the relative specific viscosity of such solutions. Thus, such studies offer the least ambiguous test of intercalation (Lerman, 1961; Satyanarayana et al., 1992; Kelly et al., 1985). Minor positive or negative changes in DNA solution viscosity are observed when binding occurs in the DNA grooves (Kelly et al., 1985). Fig. 7 shows the plot of flow time (t) of ct-DNA with increasing concentrations of TBZ. It is observed that the addition of TBZ to ct-DNA solution results in a slight increase in the flow time which is not as pronounced as observed for a classical intercalator (Vaidyanathan and Nair, 2003) and are consistent with substrates that bind to DNA through a groove-binding mode (Metcalfe et al., 2006).

CD spectra of DNA in the presence of TBZ. Conditions: [DNA] = 5.0 × 10–5 mol L−1; [TBZ] = 0–7.5 × 10−5 mol L−1.
Figure 7
CD spectra of DNA in the presence of TBZ. Conditions: [DNA] = 5.0 × 10–5 mol L−1; [TBZ] = 0–7.5 × 10−5 mol L−1.

3.10

3.10 Molecular modeling studies

Generally, molecular modeling is widely used in the design of new drugs. In recent years, this method has provided insight into the interactions between macromolecules and ligands with the help of docking programs Dock, Autodock, Surflex and so on.

The docking calculations revealed that TBZ approaches minor grooves of DNA (Fig. 8). This conclusion is consistent with the results of experimental methods and shows the power of theoretical calculations in predicting the site of interaction on DNA for a small molecule. Thiazol ring of TBZ is situated within narrow A–T (10.8 Å) regions with displacement of water molecules. Preferential binding of thiazol moiety to A–T (Filosa et al., 2009) leads to van der Waals and hydrophobic interactions with DNA functional groups same as the results of thermodynamic calculations.

Molecular docked model of TBZ with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB id: 1BNA).
Figure 8
Molecular docked model of TBZ with DNA dodecamer duplex of sequence d(CGCGAATTCGCG)2 (PDB id: 1BNA).

4

4 Conclusions

In this work, the interaction of TBZ with ct-DNA was studied by various spectroscopic and molecular modeling methods. It was revealed that TBZ is a minor-groove binder of ct-DNA. The process is spontaneous and the most important acting forces are hydrophobic along with release of water molecules. The results of experimental methods were confirmed by molecular modeling calculations.

Acknowledgment

The authors gratefully acknowledge the Research Council of Razi University for its financial support.

References

  1. , , . Liquid chromatographic fluorescence method for the determination of thiabendazole residues in green bananas and banana pulp. J. AOAC Int.. 1994;77:710-713.
    [Google Scholar]
  2. , , , , , . Binding of several anti-tumor drugs to bovine serum albumin: fluorescence study. J. Lumin.. 2009;129:541-547.
    [Google Scholar]
  3. , , , , , , , , , , . Antiparasitic drugs. IV. 2-(4′-thiazolyl) benzimidazole, a new anthelminthic. J. Am. Chem. Soc.. 1961;83:1764-1765.
    [Google Scholar]
  4. , . , , , eds. CD of Nucleic Acids in Circular Dichroism, Principles and Applications. New York: VHS; . p. :523-540.
  5. DeLano, W.L., 2004. The PyMOL Molecular Graphics System, DeLano Scientific. San Carlos, CA, USA. <http://pymol.sourceforge.net/>.
  6. , , , , , , , , . Molecular modeling studies, synthesis and biological activity of a series of novel bisnaphthalimides and their development as new DNA topoisomerase II inhibitors. Bioorg. Med. Chem.. 2009;17:13-24.
    [Google Scholar]
  7. , , , , , . Interaction of an Fe derivative of TMAP (Fe(TMAP)OAc) with DNA in comparison with free-base TMAP. Int. J. Biol. Macromol.. 2007;41:173-179.
    [Google Scholar]
  8. , , , . Determination of nucleic acids based on the fluorescence quenching of Hoechst 33258 at pH 4.5. Anal. Chim. Acta. 2006;570:21-28.
    [Google Scholar]
  9. , , , . Study on the interaction of 5-pyridine-10,15,20-tris-(p-chlorophenyl)porphyrin with cyclodextrins and DNA by spectroscopy. Spectrochim. Acta. 2007;A68:231-236.
    [Google Scholar]
  10. , . Thermodynamics of drug–DNA interactions. Arch. Biochem. Biophys.. 2002;403:1-15.
    [Google Scholar]
  11. , , , , , . Specific binding of Hoechst 33258 to the d(CGCAAATTTGCG)2 duplex: calorimetric and spectroscopic studies. J. Mol. Biol.. 1997;271:244-257.
    [Google Scholar]
  12. , , . Molecular recognition of B-DNA by Hoechst 33258. Nucleic Acids Res.. 1985;13:4825-4835.
    [Google Scholar]
  13. , , , , . Thiabendazole for the treatment of trichinosis in humans. Tex. Rep. Biol. Med.. 1969;27:581-596.
    [Google Scholar]
  14. , , , , . Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism. Biopolymers. 1973;12:89-110.
    [Google Scholar]
  15. , , , . Electrochemical studies and spectroscopic investigations on the interaction of an anticancer drug with DNA and their analytical applications. J. Electroanal. Chem.. 2009;636:93-100.
    [Google Scholar]
  16. , , , , , , , , . Structural analysis of DNA and RNA interactions with antioxidant flavonoids. Spectroscopy. 2009;23:29-43.
    [Google Scholar]
  17. , , , , . Interaction of diazinon with DNA and the protective role of selenium in DNA damage. DNA Cell Biol.. 2008;27:325-332.
    [Google Scholar]
  18. , , , , . A study of the interactions of some polypyridylruthenium(II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation. Nucleic Acids Res.. 1985;13:6017-6034.
    [Google Scholar]
  19. , , . Sequence dependent energy transfer from DNA to a simple aromatic chromophore. J. Chem. Soc. Chem. Commun.. 1992;6:470-472.
    [Google Scholar]
  20. , , , , , , , . Detection of DNA adducts in declining hop plants grown on fields formerly treated with heptachlor, a persistent insecticide. Environ. Pollut.. 1995;90:409-414.
    [Google Scholar]
  21. , , . A fluorescent complex between ethidium bromide and nucleic acids: Physical–chemical characterization. J. Mol. Biol.. 1967;27:87-106.
    [Google Scholar]
  22. , . Structural considerations in the interaction of DNA and acridines. J. Mol. Biol.. 1961;3:18-30.
    [Google Scholar]
  23. , , . Study on the interaction of morphine chloride with deoxyribonucleic acid by fluorescence method. Spectrochim. Acta Part A. 2009;71:1938-1943.
    [Google Scholar]
  24. , , , . Short-circuiting the molecular wire: cooperative binding of Δ-[Ru(phen)2d ppz]2+ and Δ-[Rh(phi)2bipy]3+ to DNA. J. Am. Chem. Soc.. 1997;119:1454-1455.
    [Google Scholar]
  25. , , , , . Spectroscopic studies on the interaction of pazufloxacin with calf thymus DNA. J. Photochem. Photobiol. B. 2008;93:172-176.
    [Google Scholar]
  26. , , . Modification of a styryl dye binding mode with calf thymus DNA in vesicular medium: from minor groove to intercalative. J. Phys. Chem. B. 2012;116:5226-5233.
    [Google Scholar]
  27. , , , , , , , . A survey of EPA/OPP, a survey of EPA/OPP and open literature on selected pesticide chemicals III, mutagenicity and carcinogenicity of benomyl and carbendazim. Mutat. Res.. 2002;512:1-35.
    [Google Scholar]
  28. , , , . Studies on the interaction of extended terpyridyl and triazine metal complexes with DNA. J. Inorg. Biochem.. 2006;100:1314-1319.
    [Google Scholar]
  29. , , , . Using Auto Dock for ligand-receptor docking. Curr. Protoc. Bioinf.. 2008;8 unit 14.1–8.14
    [Google Scholar]
  30. , , , , , , , . AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J. Comput. Chem.. 2009;30:2785-2791.
    [Google Scholar]
  31. , , . Sequence specificity of 125I-labelled Hoechst 33258 in intact human cells. J. Mol. Biol.. 1988;201:437-442.
    [Google Scholar]
  32. , , . Sequence specificity of 125I-labelled Hoechst 33258 damage in six closely related DNA sequences. J. Mol. Biol.. 1988;203:63-73.
    [Google Scholar]
  33. , , . Structure of methylene blue–DNA complexes studied by linear and circular dichroism spectroscopy. Biopolymers. 1982;21:1713-1734.
    [Google Scholar]
  34. , , , , , , . DNA interactions of monofunctional organometallic ruthenium(II) antitumor complexes in cellfree media. Biochemistry. 2003;42:11544-11554.
    [Google Scholar]
  35. , , . Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry. 1981;20:3096-3102.
    [Google Scholar]
  36. , , , . Neither DELTA- nor LAMBDA- tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation. Biochemistry. 1992;31:9319-9324.
    [Google Scholar]
  37. , , , , , , , , . Determination of genotoxicity of the metabolites of the pesticides Gluthion, Sencor, Lorox, Reglone, Daconil and Admire by 32P-postlabeling. Mol. Cell. Biochem.. 1997;169:177-184.
    [Google Scholar]
  38. , , , , , , , , . Interaction between DNA and microcystin-LR studied by spectra analysis and atomic force microscopy. Biomacromolecules. 2011;12:797-803.
    [Google Scholar]
  39. , , , , . Fluorescence studies of native and modified neurophysins. Effects of peptides and pH. Biochemistry. 1979;18:1026-1036.
    [Google Scholar]
  40. , , , , , , . Study on the interaction mechanism between DNA and the main active components in Scutellaria baicalensis Georgi. Sens. Actuat. B. 2008;129:799-810.
    [Google Scholar]
  41. , , , , , , . Interaction mechanism of 2- aminobenzothiazole with herring sperm DNA. J. Lumin.. 2012;132:507-512.
    [Google Scholar]
  42. , , , , . Persistence and efficacy of thiabendazole on potatoes for control of silver scurf. J. Agric. Food Chem.. 1993;41:2156-2159.
    [Google Scholar]
  43. , . 1972. In: , ed. Proteins of Biological Fluids. Oxford: Pergamon Press; . p. :511.
    [Google Scholar]
  44. , , . DNA binding and cleavage properties of certain tetramineruthenium(II) complexes of modified 1,10-phenanthrolines – effect of hydrogen-bonding on DNA-binding affinity. J. Inorg. Biochem.. 2004;98:219-230.
    [Google Scholar]
  45. , , . Photooxidation of DNA by a cobalt(II) tridentate complex. J. Inorg. Biochem.. 2003;94:121-126.
    [Google Scholar]
  46. , , , , . A study of the binding of colloidal Fe3O4 with bovine hemoglobin using optical spectroscopy. Colloid. Surf. A. 2009;337:102-108.
    [Google Scholar]
  47. , . Complex formation between ethidium bromide and nucleic acids. J. Mol. Biol.. 1965;13:269-282.
    [Google Scholar]
  48. , , , . A novel genotoxic aspect of thiabendazole as a photomutagen in bacteria and cultured human cells. Toxicol. Lett.. 2005;158:213-219.
    [Google Scholar]
  49. , , . Fluorometric properties of the bibenzimidazole derivative Hoechst 33258, a fluorescent probe specific for AT concentration in chromosomal DNA. Choromosoma. 1974;46:255-260.
    [Google Scholar]
  50. , , , , , . Spectral studies of the binding of lucigenin, a bisacridinium derivative, with double-helix DNA. Anal. Bioanal. Chem.. 2002;373:163-168.
    [Google Scholar]
  51. , , , , . Study of interaction of a fluorescent probe with DNA. J. Lumin.. 2009;129:1286-1291.
    [Google Scholar]
  52. , , , . Metallo-intercalators and metallo-insertors. Chem. Commun.. 2007;44:4565-4579.
    [Google Scholar]
  53. , , , , , . The mechanism of carbofuran interacts with calf thymus DNA. Spectrosc. Spect. Anal.. 2005;25:739-742.
    [Google Scholar]
  54. , , , , . Molecular spectroscopic studies on the interaction of ferulic acid with calf thymus DNA. Spectrochim. Acta Part A Mol. Biomol. Spectrosc.. 2013;112:78-83.
    [Google Scholar]
  55. , , . Phenotypic characterization of the binding of tetracycline to human serum albumin. Biomacromolecules. 2011;12:203-209.
    [Google Scholar]
  56. , , , , , . Spectroscopic and molecular modeling methods to investigate the interaction between 5-hydroxymethyl-2-furfural and calf thymus DNA using ethidium bromide as a probe. Spectrochim. Acta Part A Mol. Biomol. Spectrosc.. 2014;124:78-83.
    [Google Scholar]

Fulltext Views
631

PDF downloads
403
View/Download PDF
Download Citations
BibTeX
RIS
Show Sections

Suggested read for related articles: