Intercalation of diafenthiuron insecticide with calf thymus DNA: spectroscopic and molecular dynamics analysis

Abstract A series of biophysical experiments like UV-Vis, fluorescence, circular dichroism (CD), competitive displacement assays, voltammetric studies, viscosity measurements and denaturation effect and metadynamics simulation studies were performed to establish the mode of binding of diafenthiuron (DF) insecticide with calf thymus DNA (CT-DNA). Analysis of absorption and fluorescence spectra in Tris-HCl buffer of pH 7.4 indicates the formation of a complex between DF and CT-DNA and the binding constant of which is in the order of 104 M−1. Competitive displacement assay with ethidium bromide (EB) and Hoechst 33258 suggests that the most probable mode of binding of DF with CT-DNA may be via intercalation mode. The results of other experiments such as CD spectral studies, viscosity measurements and the effect of denaturation agent urea support the intercalation of DF with CT-DNA. Thermodynamic parameters (ΔH o, ΔS o and ΔG o) reveal that hydrogen bonds (H-bonds) or van der Waals (vdW) force is the main binding force in the spontaneous interaction between DF and CT-DNA. Molecular dynamics (MD) simulation studies confirmed the intercalation of DF into the base pairs of CT-DNA. Communicated by Ramaswamy H. Sarma


Introduction
Diafenthiuron, 1-tert-butyl-3-(2,6-di-isopropyl-4-phenoxyphenyl)thiourea (Figure 1), is an insecticide as well as an acaricide. It acts in insects as a mitochondrial adenosine triphosphate (ATPase) inhibitor. It is not persistent in soil systems but very persistent in aquatic systems. It has low mammalian toxicity but is moderate to highly toxic for most biodiversity including aquatic life, bees and worms. Stanley et al. have investigated the selective toxicity of DF on nontarget organisms such as honey bees, coccinellids, chelonus, earthworms, silkworms and fish and the results indicated that the insecticide is highly toxic to silkworm, killing more than 80% of the caterpillars in 24 h, at all doses tested (Johnson et al., 2016). Sanchez-Bayo has studied the mode of action of insecticides in relation to their toxicity to non-target organism and found that DF is very toxic to fish, amphibians, zooplankton, and worms, but less toxic to vertebrates (Sanchez-Bayo, 2012). DF is a compound of low mammalian toxicity (LD 50 2068 mg/kg) and short environmental persistence (DT 50 < 1 day), however, it is either metabolized or photo-chemically transformed to the corresponding carbodiimide, which is responsible for the inhibition of ATPase in the mitochondria (Heong et al., 2011). According to the National Registration Authority for agricultural and veterinary chemicals, Australia, DF was reported to have moderate inhalation toxicity and inflammation changes in lungs of rats and dogs.
According to the World Health Organization (WHO) classification DF is slightly hazardous (Class III) technical grade active ingredient in pesticide (WHO, 2019).
Though insecticides have a lower toxicity against lower vertebrates, heavy applications in agricultural areas may have impact on non-target organisms. The insecticides and their dislodgebale foliar residues are to be routinely monitored because of safety and health concerns of farm workers and consumers. They are one class of the chemical matters of concern, due to their probable effects of carcinogenicity, teratogenicity, and mutagenicity. These Geno toxicants have the capacity to interact with the main cellular target DNA, which can alter the structure or breakage of phosphodiester linkages within the DNA molecule (Jose et al., 2011;Kashanian et al., 2012). Thus the study of interaction of these toxicants with DNA has been the focus of some recent research in the scope to develop the mechanism of action in life science, chemistry and clinic medicine arena. Though there are no scientific reports on poisoning of DF in humans, there are news in magazines and daily newspapers related to pesticide poisoning (wherein DF is one of the active ingredients) which caused number of deaths in several parts of India (supplementary material Figure S1). Therefore, it is necessary to know the mechanism of binding of DF with DNA to understand the risky mechanism of DF in the human body to protect the farm workers and consumers. Hence, the understanding of the mechanism of interaction of DF with DNA is important and hence the present study.
The main objective of the present endeavour is to investigate the interaction of DF with CT-DNA at physiological conditions using spectral techniques such as UV À Vis, fluorescence, and CD combined with urea quenching studies, competitive displacement assays, viscosity measurements and voltammetric studies. The binding mode of DF to CT-DNA was also predicted from molecular docking and MD simulation methods to further validate the results of above mentioned spectral and analytical studies.

Experimental section
All the chemicals used in this study are commercially available analytical grade. DF and ethidium bromide (EB) were purchased from Sigma Aldrich, India. The CT-DNA was obtained from Genie, India and its purity was checked by monitoring the ratio of absorbance at 260/280 nm. The stock solution of DF (1 mM) was prepared using absolute ethanol. The stock solutions of urea, EB and Hoechst 33258 were prepared using Millipore water. Freshly prepared solutions were used for the spectral measurements. All the solutions used in the experiments were adjusted with 10 mM Tris-HCl buffer solution (pH 7.4). The details of various instruments used in the study are given in the Supporting Information.

Instrumentation details
The UV-Vis absorption spectra of DF and CT-DNA were recorded in the wavelength range of 220-600 nm carried out in Tris-HCl buffer solution by using a JASCO (V630), Japan, double beam spectrophotometer equipped with 1 cm quartz cuvettes at room temperature. The fluorescence spectra of DF were recorded in JASCO (FP 8500), Japan, spectrofluorimeter at three different temperatures viz. 298, 308 and 318 K. The circular dichroism spectra were recorded using a JASCO (J810), Japan, spectrometer at a wavelength range of 220 to 310 nm in Tris-HCl buffer (pH 7.4) at room temperature. The optical chamber of the CD spectrometer was deoxygenated with dry nitrogen before use and kept in a nitrogen atmosphere during experiments. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were carried out using CHI-643B, Austin, TX, electrochemical workstation with GC as working electrode. Pt wire and Ag as, reference and auxiliary electrodes in 10 mM Tris-HCl buffer (pH 7.4).
Viscosity measurements were carried out, using Ostwald viscometer (Made in India), in a thermostat at 25 C by keeping the CT-DNA concentration constant and varying the concentration of DF and EB. The mean values of three replicated measurements were used to evaluate the relative viscosity of the samples.

Metadynamics simulation protocol
The DF/DNA complex was first soaked in an orthorhombic water box containing 11,810 water molecules (TIP3P water model), all atom force field (OPLS-2005) and 14 chloride ions were added to neutralize the system. The energy minimization and simulation (for 100 ns) were carried out at 300 K and 1 bar pressure. During the course of simulation snapshots of the complex were taken at every successive 100 ps and saved. The two collective variables used for metadynamics simulation are: (i) distance between the centre of mass (COM) of four bases of DNA and the COM of DF and (ii) the angle between the fixed vector AB and vector AC, the distance between the COM of four bases of DNA and that of DF. Finally, the setup was minimized and pre-equilibrated using the default relaxation routine.

Absorption spectral studies
The changes in absorption spectra of CT-DNA in the presence of increasing amounts of ligands are the most common way to evaluate the interaction between them. In general, the absorption spectra of CT-DNA bound to ligands show significant hypo-or hyper-chromism and shifts in the absorption maximum due to the interaction between them. The UV-Vis spectra of free CT-DNA (95 mM) and its complexes with varying concentration of DF (0-5 mM), recorded in Tris-HCl buffer (pH 7.4), are depicted in Figure 2. It is evident from the figure that an increase in concentration of DF caused hyperchromic effect with addition of small shift in wavelength (2 nm) due to complexation with CT-DNA. Such an increase in absorbance is attributed to intercalation of DF

Fluorescence spectral studies
A quantitative analysis of the binding of DF with CT-DNA was carried out by fluorimetric titration. Fluorescence spectra of DF (25 mM) in the absence and presence of varying concentrations of CT-DNA (0-20 mM) were recorded in Tris-HCl buffer (pH 7.4) at three different temperatures ( Figure 4 and supplementary material Figures S3 and S4). As seen from the figures, DF showed a maximum fluorescence at 512 nm when excited at 256 nm. The observed larger Stoke's shift may be due to the slight conformational changes observed in the ground and excited state structures of the DF molecule (supplementary material Figure S2) (Horvath et al., 2015). A smooth decrease in fluorescence intensity of DF, without any shift in the emission maximum, took place with an increase in the concentration of CT-DNA, suggesting that DF entered into the base-pairs of CT-DNA forming a complex (Husain et al., 2013Shahabadi et al., 2017;Zhang et al., 2013).
The mechanism of quenching of the fluorescence of DF by the CT-DNA (either dynamic or static quenching) was delineated by analysing the fluorescence titration data using the following Stern-Volmer equation where F 0 and F are the fluorescence intensities of DF in the absence and presence of the quencher CT-DNA, respectively. The Stern-Volmer quenching constant K SV was determined from the linear plot of F 0 /F against [Q] (supplementary material Figure S5). The K SV values are found to be 5.6 Â 10 4 , 4.7 Â 10 4 , 4.2 Â 10 4 M À1 at 298, 308 and 318 K, respectively. The decrease in K SV values with increasing temperature indicated that the fluorescence quenching of DF by CT-DNA was static (Chandrasekaran et al., 2014;Sarwar et al., 2015;Sameena & Enoch, 2013). If static binding occurred between the ligand and CT-DNA, then the binding capability of the biomolecule at each binding site would be equal. The binding constant K b in such case can be estimated using the fluorescence titration data with the help of the following modified Stern-Volmer equation (Ling et al., 2008;Sameena & Enoch, 2013).
where F 0 and F are the fluorescence intensities of DF in the absence and presence of the quencher CT-DNA, respectively. The value of K b was obtained from the intercept of the linear plot of log [(F 0 /F)/F] vs log [CT-DNA] (supplementary material Figure S6). The K b values thus computed are found to be 3.3 Â 10 4 , 0.91 Â 10 4 and 0.10 Â 10 4 M À1 at 298, 308 and 318 K, respectively. The observed decrease in the K b values, in accordance with K SV values, with increasing temperature reiterated that the quenching of fluorescence of DF by CT-DNA is via static quenching mechanism. Also, such a static quenching may be due to the formation of complex between DF and CT-DNA in the ground state (Lakowicz, 2006). The magnitude of the K b values, in the present study, is comparable with that of ligands that intercalate into CT-DNA reported earlier (Cao & He, 1998;Keswani et al., 2021).

Thermodynamic parameters
The foregoing results of the optical spectroscopic studies indicated that DF forms a complex with CT-DNA. The nature of the interaction that play a major role in the binding of DF to CT-DNA can be established using thermodynamic parameters such as changes in free energy (DG ), enthalpy (DH ) and entropy (DS ). These values were calculated from the temperature dependent K b values using the following van't Hoff and Gibbs-Helmholtz equations ( The enthalpy and entropy changes were calculated from the linear van't Hoff plot (supplementary material Figure S7). The values of DH , DS and DG for the binding of DF to CT-DNA are found to be -0.13 kJ mol À1 , -356 J K À1 mol À1 and -26.5 kJ mol À1 , respectively. The negative sign of the free energy change indicated that the binding process was spontaneous. The negative values of both enthalpy and entropy changes indicated that H-bonding or van der Waals forces played main role in the formation of the complex between DF and CT-DNA to hold the ligand within the biomolecule in the intercalation process (Mukherjee & Singh, 2017;Zare-Feizabadi et al., 2021).

Competitive binding experiments
EB and Hoechst 33258 displacement assays were performed to establish the mode of binding of DF with CT-DNA as reported earlier . The EB/CT-DNA complex containing 3 mM of EB and 27 mM of CT-DNA was excited at 250 nm, which showed an intense emission at 601 nm. The fluorescence spectra of the complex were recorded by titrating with increasing concentration of DF (0-50 mM). In EB displacement assay, any ligand that intercalates into the CT-DNA helix will replace EB from the EB/CT-DNA complex and result in quenching of the fluorescence of the complex. As seen from Figure 5, upon subsequent addition of DF, the fluorescence of EB/CT-DNA complex was completely quenched, indicating that DF binds to CT-DNA through intercalative mode similar to the typical intercalator EB. In order to ruled out the possibility of groove binding mode of interaction between DF and CT-DNA, Hoechst 33258 displacement assay was also carried out. The concentration of Hoechst 33258 and CT-DNA was 3 mM and 27 mM, respectively and the resulted Hoechst 33258/CT-DNA complex was titrated with varying concentration of DF from 0 to 100 mM. The fluorescence titration was carried out by exciting the Hoechst 33258/CT-DNA complex at 343 nm, which exhibited emission at 468 nm ( Figure 6). As evident from the figure that, upon addition of DF to the Hoechst 33258/CT-DNA complex there was no significant quenching in the fluorescence of the complex, suggesting that DF doesn't bind to CT-DNA via groove binding mode. Thus, it may be concluded that DF binds to CT-DNA via intercalation mode .

Circular dichroism spectral study
The CD spectrum of free CT-DNA (200 mM) exhibited the characteristic CD signals in the UV region (Figure 7) viz. one positive band at 278 nm for the base stacking and one negative band at 245 nm because of the right-handed B-form helicity. Under the same experimental conditions, DF was non-signal. It was observed that, upon addition of one equivalent of DF to CT-DNA, the intensity of the negative band increased with a blue-shift to 243 nm and that of the positive band decreased without any significant shift in the peak position. A clear isoelliptic point is visible at 278 nm. These significant alterations in the base stacking and helical nature of the CT-DNA upon binding with DF indicated that the mode of binding of DF with CT-DNA is intercalation rather than electrostatic or groove binding (Monnot et al., 1992;Shahabadi & Maghsudi, 2014;Zhang et al., 2011).

Voltammetric studies
Electrochemical method was also applied to complement the proposed mode of interaction between DF and CT-DNA. Cyclic voltammogram of DF (50 mM) was recorded in Tris-HCl buffer (pH 7.4). The voltammogram of DF at a scan rate of 50 mV/s showed a cathodic peak at -0.712 V due to the reduction of DF and no anodic peak was observed in the reverse scan indicating that the electrode process is irreversible (Figure 8). The cyclic voltammograms recorded at varying scan rates from 50 to 250 mV/s are also depicted in the Figure 8. A plot of peak current versus scan rate was found to be linear (supplementary material Figure S8; r 0.997) suggesting the electrode process is an adsorption-controlled process. Further, the shift in peak potential towards more negative potential with an increase in scan rate confirmed that the electrode process in an irreversible process (Asaadi & Hajian, 2017;Jalali & Dorraji, 2012).
The nature and magnitude of the interaction between DF and CT-DNA were investigated using a relatively more sensitive differential pulse voltametric (DPV) technique. The DP voltammograms of DF (25 mM) obtained in Tris-HCl buffer of pH 7.4, in the absence and presence of CT-DNA (0-18 mM), are shown in Figure 9. It is evident from the voltammograms that the peak potential shifted to more negative potential with an increase in the concentration of CT-DNA reiterating that DF binds to CT-DNA through intercalation mode. Further, the observed decrease in peak current with an increasing in the concertation of CT-DNA may be due to the formation of DF/CT-DNA complex, which resulted in a decrease in the apparent diffusion coefficient of the electroactive species (Radi et al., 2013;Shahzad et al., 2019). The formation constant of the DF/CT-DNA complex was determined using following equation (Ahmadi & Jafari, 2011).
where K f is the formation constant, A is the proportional constant and i o and i are the peak currents in the absence and presence of CT-DNA, respectively. The value of K f was calculated from the linear plot of (i o /i oi) versus 1/[CT-DNA] (supplementary material Figure S9; r 0.997) and was found to be 1.8 Â 10 5 M À1 .

Viscosity studies
Measurement of viscosity of CT-DNA with continuous addition of a given ligand is one of the most effective conclusive methods to ascertain the mode of binding between them. It    is well-known that classical intercalators would separate the base pairs and consequently lengthens DNA helix resulting in significant increase in viscosity of DNA. On the other hand, electrostatic or groove binding ligands would decrease or show a little effect on the viscosity of the biomolecule. As shown in Figure 10, upon addition of increasing amounts of DF to CT-DNA the relative viscosity of CT-DNA increased significantly and the increase was comparable to that caused by the typical intercalator EB, indicating that DF binds to CT-DNA through intercalative mode of binding Chen et al., 2019;.

Denaturation effect
The foregoing results and discussion revealed that CT-DNA accommodates the intercalated DF molecule in the helix. Chemical denaturants such as urea can destabilize the double helix of the CT-DNA molecule, thus release the intercalated ligand and consequently alter the fluorescence behaviour significantly. To provide additional support for the intercalative mode of binding of DF to CT-DNA, the effect of added urea on the fluorescence of DF/CT-DNA complex has also been carried out. As shown in Figure 11, fluorescence intensity of the DF/CT-DNA complex decreased appreciably with continuous addition of urea (0-1 mM), indicating that the binding mode of DF with CT-DNA is through  intercalation. However, incremental addition of urea has no effect on the fluorescence of DF (supplementary material Figure S10) (Qais et al., 2017;Zhou et al., 2014).

Salt effect
Studying the effect of ionic strength on ligand-DNA interaction is also a resourceful method to analyse the binding mode between small molecules and DNA. Strong electrolytes such as NaCl are used for this purpose. It is apparent that both intercalative binding and groove binding are closely related to the DNA double helix, but the electrostatic binding can take place from outside the helix. If any molecules bound to the DNA via electrostatic manner, it reflected on the fluorescence intensity of small molecules-DNA system. In the present study, with an increasing concentration of NaCl (0-1 mM), fluorescence intensity of the DF/CT-DNA complex remained unaffected (supplementary material Figure S11). This observation confirmed that DF does not bind to the DNA via electrostatic mode (Kumar et al., 1993.

Metadynamics simulation studies
Molecular docking and MD simulation studies were carried out to envisage the preferred binding site of DF on DNA. Autodock 4.2 software was used to perform the docking studies Zhou et al., 2014). For which a typical setup containing three-dimensional structural coordinates of DNA (PDB ID: I BNA) with enclosed grid having 0.6 Å spacing was employed and all other parameters with default settings given by the software. Energetically most favourable confirmation of the DE/DNA complex, from the docking study, was used in metadynamics simulation using Desmond in Schrodinger Suite as reported by us earlier (Ponkarpagam et al., 2021). The details about the protocol adopted for the simulation are given in Supporting Information.
The atomic level interaction of DF with DNA was predicted using MD simulation studies. The simulated structure of the DF/DNA complex is depicted in Figure 12(a). It is evident from the figure that the phenyl ring of the DF molecule was stacked into the base pairs of DNA molecule. This is in good agreement with the conclusion made in various biophysical studies viz. intercalation of DF into the base pairs of DNA. The types of interactions in the DF/DNA complex were analysed (Figure 12(b)). As seen from the figure, the phenyl ring of DF binds with DG10 residue through p-p stacking (represented as blue line) and p-cation interaction (represented by green line) in addition to water mediated H-bonds. These H-bonds are omitted for want of clarity. The numbers of these p-p stacking and p-cation interactions present during the simulation period are collected in Figure 13, indicating the binding of DF with the biomolecule. Such a stacking interaction of the phenyl ring of DF might cause the significant alterations in the base stacking and helical nature of the DNA upon binding with DF as observed in the CD spectral studies. As seen from Figure 14, with the addition of DF, the root-mean-square displacement (RMSD) of DNA increased initially suggesting that the structure of the DNA gets perturbed by DF and after the formation of DF/ DNA complex the RMSD value remained almost constant throughout the simulation period (Grasel et al., 2015). Thus, the foregoing experimental observations indicated that DF intercalates into the base pairs of DNA molecule through the phenyl ring and this conclusion is strongly supported by the metadynamics simulation studies, wherein we could visualize the mode of binding of the insecticide with the biomolecule.

Conclusions
The interaction between diafenthiuron (DF) insecticide and CT-DNA was investigated using different biophysical experimental techniques and MD simulation studies. The following are the important results of the study to delineate the mechanism of the interaction between DF and CT-DNA.
i. UV-Vis and fluorescence spectral results indicated formation of a complex between DF and CT-DNA. ii. In competitive displacement assays, DF displaced EB completely from EB/CT-DNA complex, while produced no significant effect on Hoechst 33258/CT-DNA complex, suggesting that the mode of binding of DF with CT-DNA is via intercalation mode. iii. Alteration in the 278 and 245 nm bands of CD spectrum of CT-DNA upon binding with DF supported the intercalation of DF with CT-DNA. iv. Shift in electro-reduction peak potential of DF to more negative value upon binding with CT-DNA reiterated the proposed intercalation mode of binding. v. The relative viscosity of CT-DNA increased appreciable with an increase in concentration of DF and the increase is comparable with that caused by the classical intercalator EB, confirming the intercalation of DF with CT-DNA. vi. Quenching of fluorescence of DF/CT-DNA complex by the denaturation agent urea also supported this binding mode. vii. DF intercalates into the base pairs of CT-DNA with binding constant on the order of 10 4 M À1 . viii. Negative free energy change revealed that the formation of DF/CT-DNA complex is a spontaneous process. DH o < 0 and DS o < 0 suggested that the main force acting between the partners in the complex is H-bond or van der Waals force. ix. The MD simulation analysis is presented here for the intercalation of DF into the base pairs of DNA which is in good agreement with the experimental studies.
It is believed that the knowledge gained in the present study on the intercalation of diafenthiuron insecticide with CT-DNA would shed some light on the toxicity of the compound, as and when required.

Funding
The author(s) reported there is no funding associated with the work featured in this article.