Interaction of capsaicin with calf thymus DNA: A multi-spectroscopic and molecular modelling study

International Journal of Biological Macromolecules 97 (2017) 392–402

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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac

Interaction of capsaicin with calf thymus DNA: A multi-spectroscopic and molecular modelling study Faizan Abul Qais a , K.M. Abdullah b , Md. Maroof Alam b , Imrana Naseem b , Iqbal Ahmad a,∗ a b

Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh, 202002, India Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, 202002, India

a r t i c l e

i n f o

Article history: Received 29 November 2016 Received in revised form 2 January 2017 Accepted 4 January 2017 Available online 16 January 2017 Keywords: DNA binding Docking Minor groove binding Capsaicin

a b s t r a c t Studying the mode of interaction between small molecules and DNA has received much attention in recent years, as many drugs have been reported to directly interact with DNA thereby regulating the expression of many genes. Capsaicin is a capsaiciniods family phytocompound having many therapeutic applications including diabetic neuropathy, rheumatoid arthritis, prevention of DNA strand breaks and chromosomal aberrations. In this study, we have investigated the interaction of capsaicin with calf thymus DNA using a number of biophysical techniques to get an insight and better understanding of the interaction mechanism. Analysis of UV–vis absorbance spectra and fluorescence spectra indicates the formation of complex between capsaicin and Ct-DNA. Thermodynamic parameters G, H, and S measurements were taken at different temperatures indicated that hydrogen bonding and van der Waal’s forces played major role in the binding process. Additional experiments such as iodide quenching, CD spectroscopy suggested that capsaicin possibly binds to the minor groove of the Ct-DNA. These observations were further confirmed by DNA melting studies, viscosity measurements. Molecular docking provided detailed computational interaction of capsaicin with Ct-DNA which proved that capsaicin binds to Ct-DNA at minor groove. Computational molecular docking also revealed the exact sites and groups to which capsaicin interacted. © 2017 Elsevier B.V. All rights reserved.

1. Introduction A living organism has genetic material containing the coded information for its functioning that makes the DNA an obvious target of study for many researchers [1,2]. The drug-DNA interaction is an important area since it provides valuable information in the development of drugs and controlling gene expression [3–5]. DNA is also target molecule for many drugs including those under advanced clinical trials, especially anticancer drugs [6,7]. Small molecules like drugs interact with DNA via mainly three different binding modes: intercalation, groove binding and ionic interactions [8]. Many such molecules may directly interact with DNA and the factors controlling these interactions are still not very well understood. Studying these interactions has become simpler due to the availability of well-known three-dimensional structure of DNA, availability of genomic sequence and many automated computer

Abbreviations: Ct-DNA, calf-thymus DNA; AO, acridine orange; EB, ethidium bromide; KI, potassium iodide; CD, circular dichroism. ∗ Corresponding author. E-mail address: [email protected] (I. Ahmad). http://dx.doi.org/10.1016/j.ijbiomac.2017.01.022 0141-8130/© 2017 Elsevier B.V. All rights reserved.

programs. Studying these interactions also enables us to understand the mechanism of action of drugs at molecular level. Capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide) and dihydrocapsaicin constitute upto 90% of total capsaiciniods in which capsaicin accounts for approximately 71% [9]. Capsaicins have many therapeutic applications including those in diabetic neuropathy and rheumatoid arthritis [10]. This is one of the important dietary phytocompound having not only anticarcinogenic effects [11], but has also been reported to prevent DNA strand breaks and chromosomal aberrations [12]. Studies have revealed that capsaicin inhibits the activity of ethylmorphine-N-demethylase and many drug metabolizing enzymes of liver by interacting with cytochrome P-450 [13]. On the contrary, it is also evident that capsaicin has tumor promoting effects and people consuming large amount of chilli peppers are more prone to get gastric cancer [14,15]. Such compound needs to be investigated to get insights into their molecular mechanism of interaction with DNA. Therefore, in the present study, we have tried to explore the mode of interaction between capsaicin and Ct-DNA by using various biophysical and molecular modelling techniques. In silico molecular modelling complemented the in vitro interactions results and confirmed binding mode as minor groove binder.

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2. Material and methods 2.1. Materials Calf thymus DNA (Ct-DNA), Capsaicin, Hoechst 33258 and acridine orange (AO) were purchased from Sigma Aldrich, USA. Ethidium bromide (EB) was purchased from Himedia, India. All the other chemicals used were of analytical grade and were used without further purification. 2.2. Sample preparation Stock solution of Ct-DNA was prepared by dissolving 60 mg of Ct-DNA in 30 mL of 10 mM Tris–HCl buffer (pH 7.4) overnight at 4 ◦ C with gentle mixing to make a homogenous solution. Final concentration of the stock solution of Ct-DNA was measured by spectrophotometer using molar excitation coefficient of 6600 M−1 cm−1 [16]. The purity of the Ct-DNA solution was determined by calculating the absorbance ratio A260 /A280 which was found to be 1.83, showing that the DNA was free from protein and pure for experimental use. Stock solution was stored at −20 ◦ C for further use. 3 mM stock solution of capsaicin was made in absolute ethanol and diluted in 10 mM Tris–HCl buffer (pH 7.4) for further use.

393

capsaicin-DNA complex [17,18]. All the measurements were done at 25 ◦ C. The fluorescence emissions for all the solutions were recorded. The concentration of capsaicin and Ct-DNA was varied while sum of their concentration was kept constant at 30 ␮M. The difference in the fluorescence emission intensity (F) of capsaicin in absence and presence of DNA was plotted as a function of mole fraction of capsaicin. The break point of the above mentioned plot yielded the mole fraction of the bound capsaicin in the complex. The stoichiometry was obtained in terms of DNA-capsaicin [(1 − ␹capsaicin )/␹capsaicin ], where ␹capsaicin denotes the mole fraction of capsaicin. 2.7. DNA melting experiment In DNA melting experiment, the absorbance of Ct-DNA (50 ␮M) was monitored at varying intervals of temperature from 25 to 100 ◦ C at 260 nm. The same experiment was repeated for capsaicinDNA complex (50 ␮M each) to determine the change in melting temperature (Tm ) of capsaicin-DNA complex as compared to DNA alone. EB-DNA complex was taken as positive control in which the concentration of EB and DNA was 10 ␮M and 50 ␮M respectively. The absorbance recorded in each case was plotted as function of temperature. The transition midpoint of the curve was used for determining the Tm .

2.3. UV–vis spectroscopic study 2.8. Effect of urea UV–vis spectrophotometric studies were performed on UV–vis Shimadzu 1800 UV–vis Spectrophotometer. The UV–vis spectra of free capsaicin and Ct-DNA-capsaicin complex were recorded in the wavelength range 200–500 nm. The experiment was carried out in the presence of fixed concentration of capsaicin (30 ␮M) and by titrating varying concentration (10–100 ␮M) of Ct-DNA. Base line correction was carried out using blank solution containing 10 mM Tris-HCl buffer (pH 7.4).

The mode of interaction between Ct-DNA and capsaicin was further studies studying urea induced denaturation. A fixed concentration of capsaicin-DNA complex (30 ␮M each) was titrated with increasing concentration of urea (0–1 M). The capsaicin-DNA complex was excited at 280 nm and the emission spectra was recorded from 290 to 500 nm. 2.9. Circular dichroism studies

2.4. Steady state fluorescence Fluorescence studies were performed on RF-5301PC Spectrofluorophotometer, Shimadzu Scientific Instruments, Japan having xenon flash lamp and using 1.0 cm quartz cells. Fluorescence emission spectra of capsaicin were recorded in the range of 290–600 nm after excitation at 280 nm. The change in fluorescence intensity was observed by titrating the fixed concentration of capsaicin (30 ␮M) with varying concentration of Ct-DNA (10–100 ␮M). The steady state fluorescence experiment was performed at different temperatures (298 K, 303 K and 310 K) for the evaluation of various thermodynamic parameters involved in the formation of capsaicin–DNA complex. 2.5. Potassium iodide quenching This experiment was performed in two sets. In one set capsaicin alone (30 ␮M) was excited at 280 nm and emission spectra was recorded in presence of increasing concentration of KI (10–100 mM) in the wavelength range of 290–600 nm. In another set, experiment was performed in presence of Ct-DNA (30 ␮M) and capsaicin-DNA complex was excited and emission spectra were recorded at increasing concentration of KI in 10 mM Tris-HCl (pH 7.4). Ksv values were calculated in both the cases using SternVolmer equation. 2.6. Continuous variation analysis (Job’s plot) The continuous variation method also called as Job’s plot was employed for the evaluation of binding stoichiometry of the

CD spectral studies of DNA was recorded at 25 ◦ C using JASCO spectropolarimeter (J-815) equipped with a Peltier temperature controller with accuracy of ±0.1 ◦ C to maintain a constant temperature. Far-UV CD spectra of Ct-DNA in absence and presence capsaicin were monitored with speed of 200 nm per min. The concentration of DNA was 100 ␮M capsaicin was varied to maintain the molar ratios of 1:0, 1:0.5 and 1:1. The spectra of buffer solution (10 mM Tris–HCl, pH 7.4) were subtracted from the spectra of DNA and capsaicin-DNA complex for the base line correction. 2.10. Effect of ionic strength This experiment was performed to evaluate the role of ionic strength in the formation of capsaicin-DNA complex. Briefly, capsaicin-DNA complex (30 ␮M each) was titrated with varying concentration of NaCl (10–100 mM). The excitation wavelength was 280 nm and emission was recorded form 290–600 nm in 10 mM Tris-HCl (pH 7.4). 2.11. Viscosity measurements In order to further elucidate the binding mode of capsaicin, viscosity measurement was carried out in absence and presence of Ct-DNA using an Ubbelohde viscometer suspended in water bath at 25 ◦ C. To a fixed concentration of Ct-DNA (100 ␮M), varying concentration of capsaicin (20–100 ␮M) was added and the flow time of each concentration was measured thrice using a digital stopwatch. The data was plotted as(␩/␩0 )1/3 versus ratio of capsaicin to Ct-DNA

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concentration (capsaicin/DNA), where ␩ and ␩0 are the viscosity of Ct-DNA in presence and absence of capsaicin respectively. 2.12. Competitive displacement assays Dye displacement assays using different dyes (intercalating or groove binding dye) were performed using spectrofluorophotometer. In EB displacement assay, a solution containing fixed amount of Ct-DNA (30 ␮M) and EB (2 ␮M) was titrated with increasing concentration of capsaicin (10–100 ␮M). The emission spectra of EB-DNA complex were recorded from 500 to 700 nm in absence and presence of capsaicin by exciting at 476 nm. Same experiment was performed for AO displacement by exciting AO-DNA complex at 480 nm and recording emission spectra from 500 to 700 nm. For Hoechst 33258 assay, the excitation wavelength was 343 nm and emission was recorded in the range of 350–600 nm. The concentration of AO and Hoechst 33258 in both the above-mentioned experiments was 2 ␮M and the experiments were performed in 10 mM Tris-HCl (pH 7.4). The concentration of Ct-DNA in AO and Hoechst displacement assay was 30 ␮M and capsaicin concentration was varied from 10 to 100 ␮M. 2.13. Molecular docking AutoDock-vina, the molecular docking program was used for in silico interaction study between capsaicin and DNA as it is reported to do more accurate calculations and perform faster [19,20]. The three-dimensional structure of B–DNA dodecamer d(CGCGAATTCGCG)2 was downloaded from Protein Data Bank (PDB ID: 1BNA). All water molecules were deleted as it hinders the proper docking. Non-polar hydrogen atoms were merged and Kollman charges were added using MGL Tools-1.5.6 [21]. The size of the grid was set to 58 × 72 × 112 Å with spacing of 1 Å covering the entire receptor and was then saved in pdbqt format. Structure of capsaicin [CID: 1548943] was downloaded from https://pubchem.ncbi.nlm. nih.gov in SDF format which was then converted to pdb format using Chimera 1.10.2. All other docking parameters were kept as default and the conformation with lowest energy was selected. Post docking analysis was performed using Accelrys Discovery Studio 4.5 and PyMol. 3. Results and discussion 3.1. UV–vis spectroscopy To understand the mechanism of action of small molecules such as drugs, their interaction with biological macromolecules like DNA or HSA should be studied. UV–vis spectroscopy is one of the simplest and most effective techniques employed to study the drug-DNA complex formation. Fig. 1A shows the UV–vis absorption spectra of capsaicin having a peak at 280 nm in absence and presence of varying concentration of Ct-DNA (10–100 ␮M). It is clear from the figure that with addition of Ct-DNA, there was an increase in absorption along with a shift in the peak position towards lower wavelength. Hyperchromism and the change in peak position corresponds to the involvement of various non-covalent interactions between capsaicin and DNA [22]. In order to know the strength of interaction between capsaicin and Ct-DNA, following equation was employed [23] εcap εcap 1 1 = + × A − A0 εB εB K C

(1)

where A0 and A are the absorbance of capsaicin in the absence and presence of Ct-DNA, ␧cap and ␧B are the molar extinction coefficient of capsaicin and the bound complex, C is the concentration of capsaicin and K is the binding constant.

Table 1 Stern–Volmer constant and quenching constant of the DNA-capsaicin complex at different temperatures. pH

Temp (K)

Ksv ( × 103 M−1 )

Kq ( × 1011 M−1 s−1 )

R2

7.4

298 303 310

4.46 3.80 3.33

4.46 3.80 3.33

0.9938 0.9855 0.9913

The double reciprocal plot of 1/(A − A0 ) vs. 1/C (Fig. 1B) is a linear fit. The value of binding constant was calculated from the ratio of the intercept (0.4668) to the slope (1.8244 × 10−4 M). The binding constant for the interaction of capsaicin with Ct-DNA was found to be 2.558 × 103 M−1 . We also performed further experiments to obtain the detailed mechanism of binding of capsaicin to Ct-DNA.

3.2. Steady state florescence Steady state fluorescence was also performed to further investigate the interaction between capsaicin and Ct-DNA. A fixed concentration of capsaicin (30 ␮M) was excited at 280 nm and its fluorescence emission was recorded from 290 to 500 nm that showed maxima at 315 nm. With the increasing concentration of Ct-DNA, there was a progressive quenching in the fluorescence intensity without any notable change in position of the peak suggesting the interaction between capsaicin and Ct-DNA (Fig. 2A). The decrease in fluorescence intensity was found to be directly proportional to the Ct-DNA concentration. In order to express the interactions between capsaicin and Ct-DNA quantitively, the peak fluorescence emission of capsaicin in absence and presence of Ct-DNA (F0 /F) vs concentration of Ct-DNA [Q] was plotted using Stern-Volmer equation that is presented in Fig. 2B. F0 = 1 + Ksv [Q] F

(2)

Where, F0 is the fluorescence intensity of capsaicin, F is the fluorescence intensity capsaicin-DNA complex at various concentrations, Ksv is the Stern–Volmer quenching constant that gives the efficiency of quenching by Ct-DNA. The slope of the (F0 /F) vs [Ct-DNA] plot at intercept of 1 gives the values of Ksv that were found to be in the order of 103 L mol−1 (Table 1). The Stern–Volmer plot only confirms quenching process but not its type i.e. static or dynamic quenching [24]. To further confirm the quenching process, biomolecular quenching rate constant was calculated using equation (3).

Kq =

Ksv ␶0

(3)

where ␶0 is the average lifetime of a molecule that ranges typically near to 10−8 s [25], Kq calculated from the above equation is listed in Table 1 and its value was found to be in the order of 1012 L mol−1 s−1 (Table 1). Since the value of Kq obtained was higher than the limiting diffusion rate constant, the quenching process found in our study was static rather than dynamic [26]. Another way of differentiating static quenching from dynamic quenching is by calculating the values of Ksv at varying temperatures. In our study a decrease in values of Ksv with increasing temperature confirmed that nature of quenching to be static whereas reverse trend is obtained for dynamic mode of quenching [27].

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Fig 1. UV–vis absorption spectra of capsaicin in the absence and presence of varying concentration of Ct-DNA (A) and Plot of 1(/A–A0 ) vs 1/[Ct-DNA] (B).

Fig. 2. Steady-state fluorescence spectra of capsaicin in absence and presence of varying concentration of Ct-DNA (A) and the Stern–Volmer plots for the capsaicin–DNA interaction at different temperatures (B).

Table 2 Binding constant and number of binding site for DNA-capsaicin complex formation at various temperatures.

3.3. Determination of binding constant and thermodynamic parameters The modified Stern–Volmer equation below gives the relationship between fluorescence emission intensity and concentration of a quencher [28] log

F0 − F = log K + n log [Q] F

S H + 2.303 RT 2.303 R

Temp (K)

K (×103 M−1 )

n

R2

7.4

298 303 310

6.15 4.84 3.76

1.03 1.02 1.01

0.9963 0.9831 0.9906

(4)

where n is the number of binding sites and K is the binding constant. By plotting log [(F0 -F)/F] vs log [Q], the values of K and n can be obtained from the intercept and slope respectively (Fig. 3A) The value of K and n was calculated at different temperatures and their values were found to be in order of 103 M−1 similar to the value obtained using UV–vis spectroscopy (Table 2). The thermodynamic parameters determining the nature of forces involved in the complex formation are free energy change (G), entropy change (S) and enthalpy change (H). In order to calculate various thermodynamic parameters, we employed van’t Hoff equation to calculate H and S [29]. log K = −

pH

(5)

where R is the universal gas constant (8.314 kJ K−1 mol−1 ) and T is the temperature (in kelvin). By plotting log K against 1/T (van’t Hoff plot), the value of H and S were determined form slope and intercept respectively (Fig. 3B). The change in free energy (G) at different temperature was calculated from the equation: G = H − TS

(6)

The values of all the calculated thermodynamic parameters are listed in Table 3. Numerical values and the sign of various thermodynamic parameters give valuable information about the nature of forces acting between the small molecules and macromolecules. In most of cases, there is involvement of many non-covalent inter-

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Fig. 3. Modified Stern-Volmer plot of log [(F0 -F)/F] vs log [Q] for capsaicin–DNA interaction (A) and Van’t Hoff plot (B). Table 3 Various thermodynamic parameters for DNA-capsaicin complex formation at various temperatures. pH

Temp (K)

G (kJ mol−1 )

H (kJ mol−1 )

S (J mol−1 K−1 )

R2

7.4

298 303 310

−21.59 −21.42 −21.19

−31.38

−32.84

0.9951

actions such as hydrogen bond, hydrophobic interactions, van der Waals forces and electrostatic forces [30]. The negative value of free energy (G) ensures that the binding process between capsaicin and Ct-DNA is spontaneous. Moreover, the negative values of both enthalpy change (H) and entropy change (S) indicate that the hydrogen bonds and van der Waals interactions are the two major forces involved in the formation capsaicin-DNA complex [31]. 3.4. KI quenching studies To further confirm the mode of binding of capsaicin to DNA, fluorescence quenching studies in absence and presence of Ct-DNA was done taking potassium iodide as a quencher [32]. Being negatively charged, iodide ions sufficiently quench the fluorescence intensity of small molecules in aqueous medium. DNA also carry negative charge on its phosphate backbone that effectively repels the anionic quenchers such as potassium iodide. This is the reason that small molecules that have intercalated with DNA is well protected from the approach of anionic quencher and its fluorescence intensity is not quenched [33]. In contrast, the molecules that either have groove binding or electrostatic binding are well exposed to the external environment and are easily accessible by negatively charged quenchers even in presence of DNA and their intensity is readily effected [34]. In order to find out the difference in the ability of capsaicin being quenched by potassium iodide in presence and absence of Ct-DNA, we deployed above mentioned Stern-Volmer equation- (2): F0 = 1 + Ksv [Q] F

(2)

where F0 and F are the peak fluorescence intensity in the absence and presence of the potassium iodide [Q]. The Stern-Volmer quenching constant (Ksv ) is calculated from the slope of plot [F0 /F] vs [Q] at fixed intercept of 1. In case of intercalators, there is a signif-

Fig. 4. Stern-Volmer plot for fluorescence quenching of capsaicin (30 ␮M) by KI in absence and presence of Ct-DNA.

Table 4 Variation of Ksv values for iodide quenching of capsaicin in absence and presence of Ct-DNA.

Capsaicin Capsaicin + DNA

Ksv (M−1 ) Ra

R2

4.79 4.36

0.9961 0.9973

Relative reduction in Ksv (%) 8.97

icant decrease in the Ksv value in presence of Ct-DNA as compared to the Ksv value in absence of Ct-DNA [22]. However, there is a small decrease in Ksv values for groove binders and electrostatic binders. Fig. 4 shows the Stern-Volmer plot of Iodide quenching of capsaicin both in absence and presence of Ct-DNA. Table. 4 shows Ksv values and its relative change in terms of percentage. The value of Ksv for capsaicin was found to be 4.8 M−1 while in presence of Ct-DNA was found to be 4.4 M−1 . There was only a slight decrease (8.97%) (as compared to those of intercalators) in the Ksv value showing that the mode of binding of capsaicin to DNA is groove binding.

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Fig. 5. Job plot of difference in fluorescence emission plotted against mole fraction of added capsaicin.

3.5. Binding stoichiometry (Jobs plot)

Fig. 6. Thermal melting curve of Ct-DNA in absence and presence of capsaicin. EBDNA complex was taken as positive control.

The binding stoichiometry of capsaicin with Ct-DNA was determined by using continuous variation analysis (Jobs plot) by fluorescence spectroscopy. Upon excitation at 280 nm, capsaicin showed strong fluorescence while in presence of Ct-DNA, its fluorescent intensity decreased. The plot of difference in fluorescence intensity (F) vs mole fraction of capsaicin is shown in Fig. 5. The least square fitted lines intersected at ␹capsaicin = 0.28. This value corresponds to the size of 3.28 base pairs of Ct-DNA bound per molecule of capsaicin. This value suggests that binding of one molecule of capsaicin is spanned between 3–4 base-pairs of Ct-DNA base pairs. 3.6. DNA melting studies The double helix of DNA is mainly stabilized due the presence of hydrogen bonds between purines and pyrimidines of opposite strand and base pair stacking interactions. Increasing temperature weakens these DNA double helix stabilizing bonds resulting in the separation of both strands. This phenomenon is called as thermal denaturation of DNA. The temperature at which half of the double helix is denatured into single strand is known as melting temperature (Tm) of DNA [35]. Interaction of small molecules such as drugs influences Tm of DNA. Intercalation of small molecules between DNA helix enhances the stability of DNA thereby increasing the Tm by 5–8 ◦ C. For groove binding and electrostatic mode of binding, little or no change in Tm is observed [33]. The value of Tm was determined for DNA and capsaicin-DNA complex from the plot of A/A25 as function of temperature (Fig. 6). The transition midpoint of this curve yields the value of Tm. Here A is the absorbance at a given temperature and A25 is the absorbance at 25 ◦ C. The value of Tm for Ct-DNA alone was found to be 69.6 ◦ C and for capsaicin-DNA complex, it was found to be 70.5 ◦ C. Tm for EB-DNA complex was found to be 74.6 ◦ C. The change in Tm for free DNA and capsaicin-DNA complex was 0.9 ◦ C which is very small as compared to intercalative mode of binding (EB-DNA complex). A very small change in Tm supports groove binding nature of capsaicin as evident by previous experiments. 3.7. Effect of urea The addition of urea to DNA destabilizes the double helix resulting separation of DNA strands [36]. The property of urea is exploited

Fig. 7. Fluorescence emission spectra of capsaicin in the presence of varying concentration of urea.

to study the mode of binding of drugs with DNA. On adding urea, the entrapped (intercalated) drug or small molecule is released into buffer solution that causes the alteration in the fluorescent intensity of drug while in case of non-intercalators i.e. groove binders a very little effect on the fluorescent behaviour is observed [37]. The subsequent addition of urea to capsaicin-DNA complex did not had any significant effect on the fluorescent intensity of capsaicin-DNA complex (Fig. 7). This result reveals the binding mode of capsaicin to be non-intercalative. 3.8. CD spectroscopy The changes in the conformation of DNA by interaction of drugs are also examined using CD spectroscopy [38]. A typical CD spectrum of B-form of DNA exhibits a negative peak at 245 nm and a positive peak at 275 nm. The negative peak represents the righthanded helicity of DNA and positive peak is due to the base pair stacking. Intercalation of molecules into the double helical structure of DNA significantly changes the native CD spectrum of DNA while the molecules having electrostatic and groove binding mode

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Fig. 8. CD spectra of DNA-capsaicin complex. CD spectra of Ct-DNA (100 ␮M) with varying concentration of capsaicin.

Fig. 9. Effect of NaCl on capsaicin–DNA interaction.

of interaction does not have any remarkable effect on the native CD spectrum of DNA [39]. Fig. 8 shows the CD spectra of free Ct-DNA (100 ␮M) and capsaicin-DNA complex at varying concentrations (50 ␮M and 100 ␮M). With increasing the concentration of capsaicin, there was very little change in CD spectra of Ct-DNA was observed. The slight decrease at the positive ct-DNA dichroic peak might be due to transition from the extended helical structure to more compact form which is also known as the  structure [40]. This result explains the nature of binding of capsaicin to Ct-DNA to be non-intercalative. However, this little change at 245 nm and 275 nm may be attributed to certain conformational changes that may conversion of B-from of DNA to A-from [25]. 3.9. Role of ionic strength In order to establish the role of electrostatic interaction in capsaicin-DNA complex formation we studied the role of ionic strength by increasing concentration of NaCl. In case of groove binding and intercalative binding, small molecule is in close proximity of the double helix of DNA. On contrary, electrostatic binding may take place from outside of DNA helix. If we titrate NaCl to drugDNA solution, the electrostatic interaction between the drug and DNA is weakened and drug is released from the drug-DNA complex leading to an increase in the fluorescence intensity [41]. As shown in Fig. 9, on increasing the concentration of NaCl from 0 to 150 mM, negligible change in fluorescence intensity was observed. This result suggests that there is negligible involvement of electrostatic interaction in the formation of capsaicin-DNA complex. 3.10. Viscosity measurements To further authenticate the mode of interaction between capsaicin and Ct-DNA, the viscosity measurement study was performed. This technique is very sensitive to measure changes in the length of DNA and is regarded as one of the most effective and least ambiguous method to resolve binding modes of small molecules with DNA [25]. An intercalating molecule causes a substantial increase in the viscosity of DNA solution which is caused by separation of base pairs at the site of intercalation. This increases the overall length of DNA [39]. In contrast, groove binders or molecules showing electrostatic interaction with DNA have an insignificant effect on the viscosity of DNA solution [42]. The viscosity plot

Fig. 10. Effect of increasing concentration of capsaicin on the viscosity of Ct-DNA. Concentration of DNA was kept constant while varying the capsaicin concentration.

of ␩/␩0 versus [capsaicin]/[Ct-DNA] is presented in Fig. 10. It is evident from Fig. 10 that with the increasing concentration of capsaicin to DNA, negligible change in the viscosity of Ct-DNA was detected. This experiment further reveals the non-intercalative binding mode of capsaicin to Ct-DNA. 3.11. Competitive displacement assays Competitive displacements of different known dyes whose DNA binding mode are well established are often used to establish the mode of interaction between small molecules and DNA. In competitive displacement experiment, if any drug displaces the dye already bound to DNA will have the same mode of binding as that of dye. Any remarkable change in the fluorescence intensity of dye-DNA complex upon subsequent addition of drug gives valuable information regarding the mode of binding of that drug with DNA [43]. EB is a sensitive probe that intercalates within the base pairs of DNA. EB alone shows weak fluorescence. However, the fluorescence intensity of EB increases significantly when it intercalates into bae pairs of DNA [44]. Drug having intercalative mode of binding will displace EB from the DNA resulting in the decrease in fluorescence intensity of EB-DNA complex. The extent of decrease in the fluorescence intensity of EB-DNA system is directly related to the degree of intercalation of the drug to DNA [45,46]. The fluorescence intensity

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399

Fig. 11. Competitive displacement assays. (A) Fluorescence titration of EBDNA complex with increasing concentration of capsaicin. (B) Fluorescence titration of AO-DNA complex with increasing concentration of capsaicin. (C) Fluorescence titration of Hoechst-DNA complex with increasing concentration of capsaicin. (D) Fluorescence titration of capsaicin with increasing concentration Hoechst.

Fig. 12. Stern–Volmer plot (A) and Modified Stern-Volmer plot (B) of log [(F0 -F)/F] vs log [Q] for the capsaicin–Hoechst interaction.

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Fig. 13. Molecular docked structures of capsaicin complexed with DNA. Figure represents minor groove binding of capsaicin with dodecamer d(CGCGAATTCGCG)2 (PDB ID: 1BNA).

of EB-DNA complex is shown in Fig. 11A. The subsequent addition of capsaicin to EB-DNA system did not had any significant effect on the fluorescence intensity of EB-DNA complex proving that the mode of binding of capsaicin to Ct-DNA is non-intercalative. To further rule out intercalative binding nature of capsaicin, AO displacement assay was performed. AO is planar aromatic probe that also binds to DNA via intercalative mode [47]. The fluorescence intensity of AO alone is very weak as compared to AO-DNA complex. If any drug has same mode of binding will replace AO from DNA helix resulting in decrease in fluorescence intensity. Upon addition of increasing concentration of capsaicin to AO-DNA complex, negligible change in the fluorescence intensity was observed (Fig. 11B). This result again validates that capsaicin have non-intercalative mode of binding. Hoechst 33258 is a fluorescent probe, known to have groove binding nature in DNA [48]. Hoechst shows enhanced fluorescence property when it is bound to DNA [49]. Any molecule that will have groove binding mode will replace Hoechst from DNA causing the decrease in florescence. Fig. 11C shows the fluorescence intensity of Hoechst-DNA complex in absence and presence of capsaicin. With increasing concentration of capsaicin, there was no change in the fluorescent intensity of Hoechst-DNA complex. This indicates that capsaicin is not a groove binder. However, this is against our many earlier experiments such as, KI quenching, DNA melting studies, viscosity measurement etc. where capsaicin was found to be groove binder or non-intercalator. To find out that why capsaicin did not replace Hoechst from Ct-DNA despite of being a groove binder, we performed another experiment. Increasing concentration of Hoechst (3–30 ␮M) was titrated to a fixed concentration (30 ␮M) of capsaicin which is shown in Fig. 11D. With increasing concentration of Hoechst, there was a notable decrease in the fluorescent intensity of capsaicin, suggesting the interaction of capsaicin with Hoechst. This might be the reason for negative result

Table 5 Details of various binding parameters of interaction between capsaicin and Hoechst. Ksv

Kq

K

n

1.09 × 104 M−1

1.09 × 1012 M−1 s−1

5.03 × 103 M−1

0.92

in Hoechst displacement assay. Details of binding of capsaicin to Hoechst are discussed below. Many of the experiments performed in this study such as KI quenching, DNA melting, viscosity measurements and CD spectroscopy demonstrate that the capsaicin is a DNA groove binder. To finally authenticate our experimental results, we performed dye displacement experiments using site specific probes whose binding mechanism is already well established. In drug-DNA binding studies, this experiment is regarded as confirmation to detect the binding site of small molecules. So, we have also performed these experiments and found that capsaicin did not displace either AO or EB or Hoechst. To find out why capsaicin did not displaced Hoechst in spite of being a groove binder, we performed another experiment to understand the ability of capsaicin to interact with Hoechst and found that there was a strong interaction between capsaicin and Hoechst. The values of various binding constants capsaicinDNA interaction are mentioned in Table 5. Stern-Volmer plot and modified Stern-Volmer plot for the binding of capsaicin to Hoechst is shown in Fig. 12A and Fig. 12B. It was interesting to note that the value of Stern-Volmer constant (Ksv ) for capsaicin- Hoechst interaction was found to be greater than capsaicin-DNA interaction. Similar results were obtained for quenching constant (Kq ). The value of binding constant (K) for capsaicin- Hoechst interaction was approximately same as that of capsaicin-DNA interaction. Thus, it is evident that capsaicin has strong affinity towards Hoechst. When increasing concentration of capsaicin was titrated to Hoechst-DNA

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complex, capsaicin bound directly to the Hoechst and therefore was unable to displace Hoechst. 3.12. Molecular docking Molecular docking provides a detailed insight into the ligand receptor interaction that may further support the experimental results. This technique gives enough detailed information regarding drug-DNA interaction which helps in rational drug design and discovery [50]. Molecular docking not only allows flexibility of the ligand to be modelled but also uses more detailed molecular mechanics for the calculation of binding energy of ligand-receptor interaction. Minor groove targeting of DNA by a small molecule is regarded as an important tool to recognize a specific sequence of DNA [51]. Fig. 13 shows the docked structure of capsaicin to DNA obtained by AutoDock-vina [19]. The molecular pose having minimum binding energy was analysed that was found to be located in the minor groove of DNA dodecamer having sequence d(CGCGAATTCGCG)2 . The capsaicin was located in GC rich region of DNA stabilized by many non-covalent interactions. The capsaicin DNA complex was stabilized by two hydrogen bonds each with guanine. It was also found to have hydrophobic interactions with each ring of the nitrogenous base of same adenine. The minimum binding energy of the docked capsaicin-DNA complex was −6.1 kcal/ mol which is comparable with results obtained by fluorescence spectroscopy. In spite of not possessing any net positive charge, capsaicin has high binding potential with DNA as indicated by the negative value of binding energy. Thus, the mutual complementation between multi-spectroscopic studies and molecular docking results further strengthen our experimental results that there is minor groove mode of binding between capsaicin and DNA. 4. Conclusion In the current study, we investigated the non-covalent interactions between capsaicin and Ct-DNA using various biophysical techniques and molecular docking studies. Capsaicin was found to interact with Ct-DNA through non-intercalative groove binding mode. UV-absorption spectra and fluorescence titration results confirmed the formation of complex between capsaicin and Ct-DNA. The binding constant calculated at three different temperatures was found to be significantly lower than intercalators. Various thermodynamic parameters revealed that the binding process was spontaneous involving hydrogen bond and van der Waal interactions. Non-intercalative mode of binding was confirmed by KI quenching experiment, DNA melting studies, CD spectral analysis and viscosity measurements. Involvement of electrostatic interaction was ruled out by studying the effect of ionic strength. In silico molecular docking finally strengthened our experimental data confirming that capsaicin binds to minor groove of Ct-DNA. Our study could provide more help in understanding the binding mechanism of capsaicin and related compounds with Ct-DNA and the pharmacological effects of capsaicin as well as designing the structure of new and efficient drug molecules. References [1] X.L. Li, Y.J. Hu, H. Wang, B.Q. Yu, H.L. Yue, Molecular spectroscopy evidence of berberine binding to DNA: comparative binding and thermodynamic profile of intercalation, Biomacromolecules 13 (February (3)) (2012) 873–880. [2] M.L. Örberg, K. Schillén, T. Nylander, Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly (amido amine) dendrimers, Biomacromolecules 8 (May (5)) (2007) 1557–1563. [3] Y. Shi, C. Guo, Y. Sun, Z. Liu, F. Xu, Y. Zhang, Z. Wen, Z. Li, Interaction between DNA and microcystin-LR studied by spectra analysis and atomic force microscopy, Biomacromolecules 12 (February (3)) (2011) 797–803.

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