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Experimental Probing and Molecular Dynamics Simulation of the Molecular Recognition of DNA Duplexes by the Flavonoid Luteolin Varughese Mary, Poovvathingal Haris, Mathew K. Varghese, Purushothaman Aparna, and Chellappanpillai Sudarsanakumar J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00747 • Publication Date (Web): 21 Aug 2017 Downloaded from http://pubs.acs.org on August 21, 2017

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Journal of Chemical Information and Modeling

Experimental Probing and Molecular Dynamics Simulation of the Molecular Recognition of DNA Duplexes by the Flavonoid Luteolin†

Varughese Mary ǂa, P. Haris ǂa, Mathew K. Varghesea,c, P. Aparnaa, and C. Sudarsanakumara,b*

a

School of Pure and Applied Physics, Mahatma Gandhi University, Kottayam, Kerala, 686560, India

b

Center for High Performance Computing, Mahatma Gandhi University, Kottayam, Kerala, 686560, India

c

Department of Physics, Pavanatma College, Murickassery, Kerala, India 685604

*[email protected], Tel: +91 9447141561, Fax: 0481 2730423

Footnote: ǂ First and second authors have equal contribution to work. † Supporting Information

ACS Paragon Plus Environment

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ABSTRACT Luteolin (C15H10O6) is an important flavonoid found in many fruits, plants, medicinal herbs, and vegetables exhibiting many pharmacological properties. The anticancer, antitumor, antioxidant, and anti-inflammatory activities of luteolin have been reported. The pharmacological action of small molecules depends upon its interaction with biomacromolecules. The interactions of small molecules with DNA play a major role in the transcription and translation process. In this work, we explored the energetic profile of DNA–luteolin interaction by ITC. The effect of temperature and salt concentration on DNA binding was examined by UV–Vis method. The mode of interaction was further probed by UV melting temperature analysis and Differential Scanning Calorimetry. An atomic level insight on the recognition of luteolin with DNA was achieved by employing Molecular Dynamics (MD) simulation on luteolin in complex with AT and GC rich DNA sequences. AMBER force field proves to be appropriate in providing an understanding on the binding mode and specificity of luteolin with duplex DNA. MD results suggest a minor groove binding of luteolin with DNA and the binding free energy obtained is in agreement with the experimental results.


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INTRODUCTION Deoxyribonucleic acid (DNA) is an important drug target facilitating the ability to manipulate vital processes such as gene expression and gene transcription which also form the background of many life threatening diseases. Drug molecules from natural sources are of great importance today as many clinical anticancer drugs are either from natural products or their derivatives. Many natural drug molecules are more potent than synthetic ones and they act via interacting with DNA. In this scenario, studying the interaction between DNA and drug molecules at biophysical and atomic level would aid in our understanding on the mechanism of drug action and further development of potent and sequence specific drugs.

(a) (b) Figure 1. (a) General Structure of Flavonoid (b) Structure of luteolin.

Flavonoids and their conjugates, ubiquitous in plants form a large group of natural compounds. More than 6,000 different compounds are known from this class and yet to be found. They have a common skeleton of diphenylpropanes (also referred as C6-C3-C6), consisting of two aromatic rings (ring A and B) linked through a three carbon chain that forms a pyran ring (ring C) with aromatic ring A (Figure 1a). The six major subclasses of flavonoids include the flavones, flavonols, flavanones, flavanols, anthocyanidins, and isoflavones. They are widely distributed in


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fruits, vegetable and beverages of plant origin.1,2 Flavonoids have the potential for prevention and treatment of several diseases.3–9 Luteolin (3', 4', 5, 7-tetrahydroxyflavone, Figure 1b) is a crucial member of the flavones class and has been identified in many edible plants such as carrots, peppers, celery, olive oil, peppermint, thyme, rosemary, chrysanthemum, aloe vera, oregano, lettuce, pomegranate, chocolate, cucumber, lemon, cabbage, cauliflower, fennel, spinach and green tea.10 Luteolin has multiple biological effects such as antioxidant, anti-inflammatory, antimicrobial, anticancer, and anti-allergic.10–24 The anti-inflammatory, anticarcinogenic and chemopreventive effects exhibited by luteolin is in part attributed to its antioxidant and free radical scavenging capacities.11 Luteolin defends our body from carcinogenic triggers through induction of cell cycle arrest or inhibition of tumor cell proliferation or by induction of apoptosis via signalling pathways.22 Studies reveal that luteolin potentiates the insulin action in adipocytes and reduces insulin resistance in obese adipose tissues with low-grade chronic inflammation thereby fighting against diabetes.25,26 Luteolin is reported to have sufficiently high bioavailability and low metabolism rate to allow this flavonoid to have its biological activities.27,28 In this paper we have explored the mechanism and mode of interaction between calf thymus DNA and the flavonoid luteolin through calorimetric as well as computational approaches. Furthermore we have employed spectroscopic techniques in a more detailed approach to clarify the binding mode of luteolin with ctDNA. Molecular Dynamics simulation studies provide a better perception of the binding mechanism underlined, sequence specificity, hydrogen bonds and water mediated interactions involved in the DNA–luteolin binding.


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MATERIALS AND METHODS Materials. Calf thymus DNA, luteolin and phosphate buffer solution (PBS) of pH 7.0 (Density: 1.01 g mL-1 at 20 °C, potassium dihydrogen phosphate/di-sodium hydrogen phosphate) were purchased from Sigma Chemicals and used without further purification. All the other chemicals used were of analytical reagent grade and double distilled water was used throughout the experiments. Calf thymus DNA (587 to 831 base pairs) was dissolved in phosphate buffer solution (PBS) and kept for 24 hours at 4 °C with occasional stirring to dissolve to a clear solution and marked as stock solution. The concentration and purity of ctDNA was confirmed by monitoring maximum absorbance intensity of the solution at 260 nm and 280 nm. The ratio of absorbance of DNA solution at 260 nm (A260) and that at 280 nm (A280) gives a value equal to or greater than 1.8 indicating that DNA samples were sufficiently free from protein.29

Isothermal Titration Calorimetry. Isothermal Titration Calorimetry (ITC) experiments 30 were done on a VP-ITC from Microcal (Northampton, MA, USA) to elucidate the binding parameters of luteolin with DNA. Luteolin was dissolved in DMSO (19.08%) and diluted into the desired concentration by adding PBS containing 100 mM NaCl. Equal volume of DMSO was also added to the ctDNA solution in PBS buffer (100 mM NaCl) so that the buffers for ctDNA and luteolin are identical. The ITC experiments were carried out by keeping the receptor DNA in the cell of ITC machine and injecting small aliquots of the ligand luteolin from the syringe. The concentration of the receptor is kept constant and the ligand concentration was changed. Both the receptor and the ligand solutions were degassed in a degassing station before the experiment. The cell of the ITC machine was filled with 0.01 mM ctDNA and the syringe was loaded with 0.4 mM luteolin. The ITC titration experiments were carried out by injecting the luteolin from


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the syringe to the cell containing receptor DNA at 25 °C. From the rotating syringe an initial volume of 2 µL of luteolin was injected to the cell containing DNA solution. All remaining injections were 10 µL each. The time taken for every injection was 20 s and a relaxation time of 240 s was given between two consecutive injections so that endothermic peak resulting from the reaction return to the base line. The total number of injections was 29. The stirring speed and reference power were 307 rpm and 15 µcal respectively. The ITC data were analyzed using ORIGIN software from Microcal. The data fitting was done by nonlinear least square method. A single binding site model was used for the analysis of the binding parameters. The binding constant (K), and enthalpy change (∆H) were obtained from the fitted data. The Gibbs binding free energy (∆G) and the entropic term (-T∆S) were evaluated from the standard equations, ∆ G = − RT ln K ∆G = ∆H − T∆S

(1) (2)

UV–Vis Absorption Spectroscopy. The molecular interaction of luteolin with ctDNA at different temperatures was monitored by UV–Vis experiments on a Shimadzu UV-2600 spectrophotometer equipped with a temperature controller. The absorbance of ctDNA with varying concentration of luteolin was measured at 25, 37, and 45 °C. The absorbance of luteolin was also monitored varying the concentration of ctDNA at 25, 37, and 45 °C and the binding constants were calculated. UV–Vis spectroscopic experiments were also conducted to understand the effect of ionic concentration (Na+ ions) on the binding of luteolin to ctDNA. The experiments were performed at various NaCl concentrations (50, 100, 200, 400 mM) keeping luteolin at constant concentration and varying the concentration of ctDNA. DNA Melting Technique. Shimadzu UV-2600 double beam spectrophotometer with a


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temperature controller was used to perform DNA melting temperature analysis. Luteolin was dissolved in DMSO and diluted into the desired concentration by adding PBS containing 100 mM NaCl. Equal volume of DMSO was also added to the ctDNA solution in PBS buffer (100 mM NaCl) so that the buffers for ctDNA and luteolin are identical.The absorbance intensity of ctDNA at 260 nm in the presence and absence of luteolin was monitored from 20 to 110 °C at a rate of 1 °C min-1 and plotted. The melting temperature analysis was performed for 20 µM ctDNA alone and ctDNA (20 µM)–luteolin (5, 20, and 30 µM) complexes. The melting temperature (Tm) of ctDNA was determined as the transition midpoint using the derivative method of Shimadzu Tm analysis software. Differential Scanning Calorimetry. The thermodynamic profile of ctDNA–luteolin interaction was measured by the Differential Scanning Calorimetry (DSC) in a Nano DSC (TA, Waters LLC, New Castle, USA). Luteolin was dissolved in ethanol and diluted into the desired concentration in PBS buffer (100 mM NaCl). Equal volume of ethanol was also added to the ctDNA solution so that the buffers for ctDNA and luteolin are identical. The reference and sample solutions were first degassed in a degassing station (TA, Waters LLC, Newcastle, USA) to remove bubbles before loading to the DSC machine. To set the base lines both the cells were loaded with buffer solution, equilibrated for 15 min at 20 °C, and scanned at 3 atm pressure from 20 to 110 °C at 1 °C min-1. Then the sample cell was filled with the ctDNA (300 µM), equilibrated for 15 min at 20 °C, and scanned at 3 atm pressure from 20 to 110 °C at 1 °C min-1. The experiment was repeated with ctDNA (300 µM)–luteolin (90 µM) complex. The experimental data were analyzed using the NanoAnalyze software v3.5.0 (TA, Waters LLC, Newcastle, USA).


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Computational Methods. The luteolin structure was obtained as cif data receptor DNAs, 2DND [d(CGCAAATTTGCG)2] 1CGC [d(CCGGCGCCGG)2]

34

32

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31

and the starting

, 1DNE [d(CGCGATATCGCG)2]

were taken from the protein data bank (PDB)

DNA structures were prepared using CHIMERA

36

35

33

and

. The receptor

for docking with the luteolin molecule

removing the associated ligands and crystal waters and then minimized. The flexible docking protocol of DOCK6.6 37 was used to probe the binding mode of luteolin to receptor DNA and the best scored binding mode was further refined using amberscore method

38

to yield a better

binding conformation. The DNA-luteolin complexes obtained from docking were designated as at6lut, at4lut, and cgclut for the receptors d(CGCAAATTTGCG)2, d(CGCGATATCGCG)2 and d(CCGGCGCCGG)2 respectively. The nucleic acid-luteolin complexes obtained from docking were used as initial complex conformations to set up Molecular Dynamics simulations. MD simulations were done using pmemd.cuda 39 module of AMBER14 40 suite of programs. The luteolin molecule was optimized at the HF/6-31G* level using the Gaussian 09 package

41

, electrostatic potentials (ESP) were

generated using Merz-Kollman population analysis method

42

and the partial atomic charges

were derived (Figure S1) from the ESP using RESP 43 program module of AMBER in consistent with the general AMBER force field (gaff) 44. The parameters for nucleic acids were available in the AMBER database and the Cornell

45

forcefield with ff99bsc0

46

parameters were used for

nucleic acids in the current simulations. The at6lut, at4lut, and cgclut simulations were built and run following the same protocol. The simulations were setup in the xLEaP module of AMBER. The DNA sequence in complex with luteolin was loaded into xLEaP, Na+ counterions were added to neutralize the system against the DNA backbone charges and then solvated in a truncated octahedron box of TIP3P water

47

extending to 11 Å from the solute in all directions.


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The simulation was performed with periodic boundary conditions and the electrostatic interactions were accounted with particle mesh Ewald method

48,49

. The system was minimized,

first the solvent and counterions with restraints on DNA and luteolin and then the whole system. Then the system was heated up to 300 K in 100 ps, with restraints (10 kcal mol-1 Å-2) on the DNA–luteolin complex at constant volume under temperature controlling using Langevin thermostat. Further, the system was equilibrated at constant pressure to equilibrate the density. The restraints on the DNA–luteolin complex were decreased from a value of 10 kcal mol-1 Å-2 to 5, 2.5, 1, 0.5, 0.1, and then to 0.01 kcal mol-1 Å-2 with each step of 50 ps.50,51 After this 300 ps of equilibration, a production simulation of 20 ns was carried out and atomic coordinates were saved at intervals of 1 ps. The 20 ns trajectory provides 20,000 conformations which were clustered using MMTSB 52 and the representative structure having least rms deviation from the centroid of the largest cluster was extracted for helical parameter calculations and comparisons. The rms deviation, hydrogen bonding, and grid analysis were done using ptraj. VMD

53

was used to visualize the trajectories

and figures were generated. Helical parameters of the representative structures were calculated using 3DNA 54. The graphs were plotted with the plotting program XMGRACE. The binding free energy of the Nucleic acid–luteolin complexes were calculated with the MMPBSA 55,56,57 single trajectory approach implemented in AMBER 14. The method used is same as implemented in our previous paper 57. The snapshots for the free energy analysis were extracted from a stable 3 ns region of the 20 ns production run with an interval of 10 ps which yields 300 frames. RESULTS AND DISCUSSION Isothermal Titration Calorimetry. To understand the molecular interaction of a drug with


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macromolecule, knowledge of the energetic profile of interaction is needed. A complete energetic profile of the binding of luteolin with ctDNA could be obtained from a single ITC experiment including number of binding sites, binding free energy, and the enthalpy and entropy changes at 25 °C. Luteolin was injected in equal aliquots to the ctDNA solution at constant concentration and the binding reaction was monitored. Figure 2 shows the ITC profile of the interaction of luteolin with ctDNA. Every single injection of the ligand luteolin into the solution of macromolecule ctDNA, give rise to a corresponding heat burst curve as shown in the upper panel of Figure 2. By integrating the area under each peak and subtracting the heats of dilution, the thermogram for the interaction of luteolin with DNA was obtained. The corrected heat as a function of the molar ratio of luteolin and ctDNA is presented on the lower panel of the Figure 2. The experimental data of injection heats and the calculated fit of the data are represented by solid rectangular points and solid line respectively.

Table 1. The energetic profile of the interaction of the luteolin with ctDNA from the ITC experiment at 25 °C. K ∆H -T∆S ∆G Binding sites

M-1

kcal mol-1

kcal mol-1

kcal mol-1

N1

8.63(±0.95) 106

-22.72(±0.18)

13.24

-9.48(±0.18)

N2

2.69(±0.14) 106

-23.85(±0.29)

15.09

-8.76(±0.29)

N3

2.54(±0.08) 105

-24.60(±0.49)

17.23

-7.37(±0.49)

N4

1.28(±0.05) 104

-103.7(±2.6)

98.09

-5.61 (±2.6)


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Figure 2. Representative ITC profile for the titration of luteolin into the solution of ctDNA in PBS buffer at 25 °C. In the top panel the heat burst curves are the result of successive injection of aliquots of luteolin into ctDNA. The bottom panel represents the corresponding normalized heat signals versus molar ratio.

The binding reaction of luteolin with ctDNA is exothermic in each injection. ctDNA has the possibility of multiple binding sites for luteolin as a minor-groove binder. The data were best fitted to variable binding site model and four binding sites were obtained. The values obtained for each binding site are tabulated in Table 1. The binding constant of luteolin with ctDNA is in the range 106 to 104 M-1. Favorable negative enthalpies of binding and unfavorable entropic contributions are deduced from ITC. The Gibbs free energy of binding obtained for each binding site are ∆G1= -9.48(±0.18) kcal mol-1, ∆G2= -8.76(±0.29) kcal mol-1, ∆G3= -7.37(±0.49) kcal mol-1, ∆G4= -5.61(±2.6) kcal mol-1. The thermodynamic parameters obtained from ITC indicate that the binding of luteolin with ctDNA is favorable, the reaction is exothermic and enthalpy driven and also costing in entropic contribution. The ITC values are in agreement with some


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reported values of minor groove binding drugs.57–63

UV–Vis Absorption Spectroscopy. The mode of binding and affinity of DNA with small molecules can be assessed from UV–visible absorption spectroscopy.

64–67

The interaction of

luteolin with ctDNA was studied by conducting UV–visible spectroscopic titration experiments at different temperatures and salt concentrations. The binding constant of luteolin with ctDNA is calculated upon the change in the concentration of ctDNA keeping the concentration of luteolin constant using the following host–guest equation57, 58 (3)

A0 εG εG 1 = + ⋅ A − A0 ε H −G − ε G ε H −G − ε G K lut C ctDNA

(3)

where A0 and A are the absorbance of luteolin at λmax in the absence and presence of ctDNA respectively. Similarly εG and εH−G are the molar extinction coefficients of luteolin at λ352 in the absence and presence of ctDNA, respectively. The concentration of ctDNA, CctDNA, was found using the Beer–Lambert law

CctDNA =

A0 ε D .l

(4)

where A0 is the absorbance of ctDNA at 260 nm and ε D is the molar extinction coefficient of ctDNA. l =1cm is the light path length of the cuvette. The value of binding constant (Klut) was calculated from the intercept to slope ratio of the linear plot between A0/(A−A0) and 1/[ctDNA]. Effect of Temperature on ctDNA–luteolin binding. Figure 3(a-c) shows the effect of temperature on ctDNA–luteolin binding at 25, 37, and 45 °C and the corresponding absorbance versus luteolin concentration plots are given in Figure S2. Figure 3d shows that luteolin exhibits


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two peaks at 268 nm and 352 nm in PBS buffer solution containing 100 mM NaCl and DMSO. They do not show any characteristic change except for the reduction in the absorbance intensity as temperature increases. The absorbance of ctDNA at 260 nm also does not change in this temperature range while the absorbance of luteolin in complex with ctDNA at 352 nm decreases with increasing temperature.

Figure 3. UV–Vis spectra of 100 µM ctDNA on increasing concentration of luteolin (0–44.8 µM) at (a) 25, (b) 37, and (c) 45 °C, (d) UV–Vis absorption spectra of 100 µM ctDNA, 22.8 µM luteolin, and 22.8 µM luteolin plus 100 µM ctDNA complex at 25, 37, and 45 ºC.

The effect of temperature on ctDNA–luteolin binding was again monitored at 25, 37, and 45 °C keeping luteolin at constant concentration and varying ctDNA concentration. The absorbance intensity of luteolin decreases on adding ctDNA as shown in Figure 4(a-c) C and the corresponding absorbance versus ctDNA concentration plots are given in Figure S3. The binding constant of luteolin with ctDNA at different temperatures were determined by plotting A0/(A-


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A0) versus 1/[ctDNA] graph as shown in Figure 5 and the values are given in Table 2. In all the temperatures, no isosbestic points were observed. The hypochromic effect without significant change in λmax shows that the interaction between luteolin and ctDNA is not a strong type like intercalation but a groove binding.64,67

(a) (b) (c) Figure 4. UV–Vis spectra of 34 µM luteolin on increasing concentration of ctDNA (0, 5, 10, 20, 34, 50, 100, 200, 300, and 358 µM) at (a) 25, (b) 37, and (c) 45 °C.

Figure 5. The double reciprocal graph of 1/[ctDNA] versus A0/(A–A0) at 25, 37, and 45 °C. Table 2. The binding constant of ctDNA–luteolin interaction at different temperatures. R2

Temperature ° C 25

K (10 ) M-1 8.99

0.99904

37

1.96

0.98507

45

2.21

0.96773

3


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Dependence of Na+ ion concentration on luteolin binding with ctDNA. Investigating the effect of Na+ ion concentration on the binding of a ligand to DNA is an efficient method to understand the binding mode. Varying the ionic strength has little effect on a molecule intercalated between adjacent base pairs of DNA as it is protected from the changes in the solvent environment whereas a groove binding molecule is more exposed to variations in solvent environment.68 To monitor the dependence of ion concentration on the binding of luteolin with ctDNA, UV–Vis spectroscopic titration experiments were carried out at 25 °C on a Shimadzu UV-2600 spectrophotometer. The UV–Vis absorption spectra of ctDNA and luteolin at different salt concentration and varying concentration of ctDNA are given in Figure S4 and the corresponding absorbance versus ctDNA concentration plots are given in Figure S5. The binding constant of luteolin with ctDNA at different salt concentrations were determined from A0/(A-A0) versus 1/[ctDNA] graph (Figure 6a).

(a) (b) Figure 6. (a) The double reciprocal graph of A0/(A-A0) versus 1/[ctDNA] at different salt concentrations at 352 nm (b) Concentration of NaCl versus binding constant of luteolin with ctDNA. All the data were fitted with good values of R2. On increasing Na+ ion concentration the binding constant decreases (Figure 6b) as 50 mM: 5.72103 M-1, 100 mM: 2.26103 M-1, 200 mM:


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2.19103 M-1, and 400 mM: 1.44103 M-1. Thus we may infer a groove binding of luteolin with ctDNA mainly favored by electrostatic contribution.63, 69–71

Melting Temperature Analysis. The mode of binding of a drug molecule to DNA can be investigated with different methods. Among them melting temperature Tm analysis is a prominent one. By determining the Tm of DNA and DNA–drug complexes we can infer whether the drug intercalates between the base pairs of DNA or binds to the grooves of DNA. The intercalative mode of binding stabilizes the DNA double helical structure resulting in an increase in melting temperature by about 5–8 ºC while non-intercalative binding causes less or no significant increase in melting temperature.59 Melting Temperature of ctDNA at various concentration of luteolin was measured to confirm the binding mode of luteolin with ctDNA in a concentration dependent manner. The melting temperatures of ctDNA duplexes were monitored by observing the absorbance of ctDNA and ctDNA–luteolin complexes at 260 nm over a temperature range from 20 to 110 °C at a rate of 1 °C min-1.

Figure 7. Melting temperature (Tm) analysis of ctDNA and ctDNA–luteolin system monitoring the absorbance at 260 nm (a) 20 µM DNA (b) 20 µM DNA + 5 µM luteolin (c) 20 µM DNA + 20 µM luteolin and (d) 20 µM DNA + 30 µM luteolin in PBS buffer containing 100 mM NaCl and DMSO. The path length of the cuvette used is 1 cm.


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Table 3. The melting temperature Tm of ctDNA at various concentrations of luteolin. Sl.No

System

Tm (°C)

1

20 µM DNA only

84.9±0.1

2

20 µM DNA + 5 µM luteolin

85.6±0.1

3


86.2±0.1

4


85.8±0.1

Figure 7 shows the melting profile of (20 µM) ctDNA and (20 µM) ctDNA–luteolin (5, 20, 30 µM) complexes. The value of Tm was determined by derivative method and tabulated in Table 3. From the Table it is clear that there is no significant change in Tm on increasing the concentration of luteolin. Temperature-dependent absorption of luteolin alone at 352 nm (5 µM, 20 µM, and 30 µM luteolin) is also shown in Figure S6. The concentration dependent measurement of the value of Tm thus confirms a non intercalative binding of luteolin with ctDNA. Such a small change in the value of Tm are also reported for some minor groove binders.57–59,63, 72–75 Differential Scanning Calorimetry. Differential Scanning Calorimetry (DSC) is a very sensitive technique to explore the thermodynamics of binding interaction. The thermodynamics underlying the binding of luteolin with ctDNA was studied by DSC experiments. The DSC curves for thermal melting of ctDNA and ctDNA–luteolin complex are shown in Figure 8. The melting temperature of ctDNA changes from 93.41 °C to 93.43 °C. The change in melting temperature of ctDNA upon binding with luteolin (∆Tm) is negligible and comparable with some reported values.57,58. The values of ∆Tm obtained from DSC and UV–Vis melting experiments are in good agreement. The negligible change in melting temperature of ctDNA upon binding with luteolin confirms the minor groove binding of luteolin with ctDNA.


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Figure 8. Thermal melting curves of ctDNA (300 µM) and ctDNA (300 µM)–Luteolin (90 µM) complex from DSC experiments. The results from NanoAnalyze are plotted for General method of modelling. The correction of melting curve data was done by the subtraction of the buffer blank data set.

Thus the DSC experiment provides a good support for the groove binding of luteolin with DNA. The difference in melting temperature of ctDNA obtained from DSC and UV melting studies is due to the difference in their buffer conditions.

Molecular Dynamics Simulation Study. Molecular docking and simulation could support the experimental results from biophysical studies and have further understanding of the binding mechanism involved in the DNA–drug interaction. Upon the experimental evidences that luteolin binds to the minor groove of DNA, AT and GC rich DNA sequences with well defined minor and major groove sites were prepared for docking. The docking yields minor groove as the preferred binding site and generates DNA–luteolin complexes for MD simulation. The 20 ns unrestrained MD simulations of the three minor groove complexes at6lut, at4lut and cgclut were stable as indicated by the rms deviations (Figure 9a–c). The luteolin was bound to the minor groove of the duplexes till the end of the simulation with the rms deviation of the luteolin stable


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around ~1.5, 1 and 8 Å for at6lut, at4lut and cgclut respectively. The cgclut system attains a conformation different from the initial docked pose and stabilizes around an rms deviation of 8 Å as shown in Figure 9c. The potential and kinetic energies of the trajectories were also stabilized after the equilibration run.

In at6lut, at4lut and cgclut systems all the base-pair hydrogen bonds in the duplexes were retained throughout the simulation with high occupancies with only terminal hydrogen bonds of AT rich duplexes having lower occupancies. Thus in all the systems base pair hydrogen bonds were retained showing that the duplex systems are intact throughout the simulation.

Figure 9. The rms deviations from the starting structures of the DNA (black) and luteolin (green) in (a) at6lut, (b) at4lut and (c) cgclut systems.

DNA Conformation and luteolin binding Interactions. The conformations of the DNA–luteolin complexes extracted from the 20 ns trajectories were clustered using MMTSB. Representative structures with the criteria of smallest rms deviation from the centroid were taken from each of the clusters generated and used for the calculation of helical parameters.


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In at6lut system, 2 clusters were obtained with 12109 (60.55%) conformations in the first and 7891 (39.45%) conformations in the second. The rms deviation between the representative structures of cluster 1 and 2 is 0.97 Å for the duplex and 0.62 Å for the luteolin molecule. Figure 10a shows the superposition of starting structure and the representative structures from each cluster of at6lut. The rms deviation of the representative structures from the starting structure are cluster 1: 1.58 Å / 1.16 Å and cluster 2: 1.89 Å / 1.06 Å for at6 duplex / luteolin complex. In the most

populated

cluster

1,

the

luteolin

molecule

is

bound

in

the

region,

A4•T21:A5•T20:A6•T19:T7•A18; the regions of close contact are shown in bold. Luteolin is positioned in the at6 region of the two DNA strands, parallel to the groove almost equidistant from both the sugar phosphate–backbone chains. The binding mode is almost similar in both clusters. The average rise and twist are 3.22 Å and 33.66° and the number of residues per turn is 10.7. The minor groove width is reduced to 4.78 Å in the binding region and 6.53 Å in other regions. Such a reduction in minor groove width at the binding site is a characteristic feature observed in many prominent minor groove binding DNA–ligand complexes.76–78 Sequence dependent simulation studies of DNA–curcumin

79

and DNA–piperine

57

complexes were

reported. Reduced minor groove widths of 5.5 Å and 4.8 Å were observed in the binding region of at6–curcumin and at6–piperine complexes respectively. The O7-H group of luteolin is in hydrogen bond with DNA duplex atoms. The O–HO, C–HO, and C–HN hydrogen bonds are observed (Figure 11a) between luteolin and base pair atoms of the duplex as shown in Table 4.


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(a)

(b)

(c)

Figure 10. The superposition of starting structure (magenta) and the representative structures (cluster 1: yellow, cluster 2: green) from each cluster of (a) at6lut (b) at4lut and (c) cgclut.

In at4lut system, the conformations generated were clustered into 2, with 12044 (60.22%) in cluster 1 and 7956 (39.78%) in cluster 2. The superposition of starting structure and representative structures from the clusters are shown in Figure 10b. The rms deviation between cluster 1 and 2 is 1.14 Å for the duplex and 1.11 Å for luteolin. From the starting structure, cluster 1 shows an rms deviation of 1.59 Å and 1.12 Å while cluster 2 shows an rms deviation of 1.57 Å and 0.89 Å for the at4 duplex and luteolin respectively. Luteolin is bound in the at4 region, A5•T20:T6•A19:A7•T18:T8•A17, in the center of the minor groove as in at6lut. In cluster 2 also, luteolin binds in the same region without considerable deviation. The average rise, twist and the number of residues per turn are 3.09 Å, 34.46° and 10.45 respectively in cluster 1. The minor groove width is 5.16 Å in the binding region and 7.51 Å in other regions; consistent with values reported for at4-curcumin (5.4 Å) 79 and at4-piperine (5.5 Å) 57 simulations where a


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reduced minor groove width was observed in the binding site. In the binding region, O–HO, C–HO, and C–HN hydrogen bonds (Figure 11b) are observed between luteolin and base pair atoms of the duplex as listed in Table 4.

Figure 11. Hydrogen bonds observed between the luteolin atoms and the DNA duplex atoms of (a) at6lut, (b) at4lut, and (c) cgclut systems. (d) Structure of luteolin molecule with atom names.


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Table 4: Interactions of the luteolin atoms with the duplex atoms in at6lut, at4lut, and cgclut systems. System DNA Atom Luteolin Atom Distance (Å) at6lut

DT19-O2 DT19-O2 DT20-O2 DA6-N3 DT7-O2

O7 C8 C6’ C6’ O7

2.77(0.16) 3.35(0.13) 3.43(0.11) 3.44(0.11) 2.99(0.25)

at4lut

DT6-O2 DT6-O2 DA7-N3 DA19-N3

O7 C8 C6’ C6’

2.73(0.14) 3.37(0.12) 3.50(0.08) 3.46(0.11)

cgclut

DG6-N2 DG16-N3 DC17-O2 DC5-O2 DG16-N2 DC5-O2

O3’ O3’ O7 C8 O1 O7

2.93(0.15) 2.99(0.18) 2.88(0.23) 3.37(0.13) 3.39(0.14) 2.95(0.29)

In the cgclut system, 2 clusters were obtained with 13795 (68.97%) conformations in cluster 1 and 6205 (31.03%) conformations in cluster 2. The rms deviation between the representative structures of the two clusters is 1.66 Å for the duplex and the corresponding rms deviation of luteolin is 6.32 Å (Figure 10c). In the first few nanoseconds of simulation which belong to the cluster 2, luteolin is bound towards the first stand (G6,C7,C8) quite away from the second strand. Then the complex attains a more confined conformation which forms the most populated cluster 1. Luteolin moves two base pair step towards 5' end and bound in the region G3•C18:G4•C17:C5•G16:G6•C15, aligned in between the duplex strands. The average rise and twist are 3.18 Å and 35.17° respectively and the number of residues per turn is 10.2 in cluster1. The minor groove widths are 5.79 Å and 8.22 Å near and away from the ligand respectively. Unlike cgc–piperine complex

57

cgclut has more reduced minor groove. The hydroxyl oxygen


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atoms of luteolin O3’, O7 and O1 are participating in hydrogen bonding with duplex atoms as N–HO, O–HN, C–HO, and O–HO hydrogen bonds (Figure 11c and Table 4). The backbone torsion angles α–γ were in the preferred values except for some variations which might be due to the binding of the ligand. The glycosidic torsion χ is in -ac region for all residues in all the clusters. In all the three systems at6lut, at4lut, and cgclut the sugar puckers were mainly distributed among C2' endo and C1' exo, both of which belong to the B-form family. O4' endo sugar pucker which is the lowest energy intermediate between A and B forms were also seen; this is also reported in the starting crystal structures.

Solvation and water mediated binding interactions. Water molecules play a significant role in the binding of the luteolin molecule to the DNA duplex. The representative structure belonging to the largest cluster in each system is extracted and the water mediated contacts between the ligand and the duplex were analyzed.

(a)

(b)

(c)

Figure 12: Representative structure from the most populated cluster1 with first shell of water (a) at6lut (b) at4lut and (c) cgclut. The water network in the minor groove is found to be adjusted to incorporate the luteolin molecule. Luteolin exhibits water mediated interactions with DNA through two or more water


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molecules. Oxygen atoms O3’, O4’, O4, O5, and O7 of luteolin are involved in the water mediated interactions with high occupancies ranging from 170 % to 63 % and in the distance range of 2.99 to 3.06 Å. The water mediated hydrogen bonds observed in the three systems ranges from 1.60 Å to 3.01 Å (Figure 12a–c). Water molecules are reported to have important role in the minor groove recognition of DNA by small molecules.

80

MD simulation studies on

DNA–DAPI minor groove complexes also report such water mediated interactions57,79,81. Hydration of the DNA-luteolin complexes were examined with grid analysis facilities of ptraj module. Figure 13(a-c) shows the hydration pattern of at6lut, at4lut, and cgclut complexes contoured at ~2 times bulk water density. The phosphates and groove regions of the duplexes were hydrated except for the luteolin bound region in the minor groove as expected. The UV–Vis spectroscopic studies show the influence of Na+ ions on the binding of luteolin to ctDNA. In our MD simulation studies we have added Na+ ions as counterions to neutralize the system and alleviate the electrostatic repulsion among negatively charged phosphate groups of the DNA.82,83 Figure 13(a-c) shows the Na+ ion density (in yellow) around DNA. Na+ ions distribution were found in the minor groove regions where the ligand is not present and there is a high Na+ density region in the proximity of the carbonyl oxygen atom O4 of the luteolin molecule which may influence the ligand binding. Na+ ion mediated interactions between luteolin and DNA were not observed while water molecules were found to take part in such interactions. 82,83


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(a) (b) (c) + Figure 13: Hydration of water (red) and Na ions (yellow) around (a) at6lut, (b) at4lut, and (c) cgclut systems with water density contoured at ~2 times bulk water density.

Free Energy Calculations. MM-PBSA free energy analysis was done to account the structural stability of the DNA–luteolin complexes. The values of binding affinities calculated are tabulated in Table 5. In all the systems van der Waals and electrostatic contributions from the molecular mechanical component (EMM) are found to be favorable for the DNA–luteolin binding. The non-polar part of the solvation free energy is favorable but the polar part is highly unfavorable, resulting in an unfavorable total solvation free energy. NMODE calculations give negative values for the solute entropic contribution which indicates a reduction in receptor and ligand configurational freedom upon complexation. NMODE calculations overestimate the rotational/translational entropy upon binding.

However the binding free energies between

luteolin and DNA duplexes which include the solute entropic contribution are appropriate for our work.


26

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Table 5. Binding energy components of at6lut, at4lut, and cgclut complexes. All the values are in kcal mol-1. at6lut

at4lut

cgclut

∆EELE (a)

-26.25(0.40)

-34.69(0.27)

-29.86(0.21)

∆EvdW (b)

-37.93(0.14)

-37.75(0.15)

-34.70(0.16)

∆EMM (c=a+b)

-64.18(0.43)

-72.43(0.26)

-64.26(0.22)

∆Gpol (d)

45.83(0.46)

55.65(0.24)

45.42(0.18)

∆Gn-pol (e)

-4.02(0.01)

-4.05(0.01)

-3.80(0.01)

∆GSOL (f=d+e)

41.81(0.46)

51.59(0.24)

41.63(0.17)

∆PBTOT (g=c+f)

-22.37(0.15)

-20.84(0.17)

-22.63(0.17)

∆TSTOT (h)

-15.44(0.54)

-15.26(0.50)

-17.02(0.50)

∆G (g-h)

-6.93(0.69)

-5.58(0.67)

-5.61(0.67)

The total binding free energy obtained for at6lut is -6.93 kcal mol-1, for at4lut it is -5.58 kcal mol-1 and cgclut has the value -5.61 kcal mol-1, including the unfavorable entropic contribution. These values are consistent with binding free energy value obtained from ITC. From the results it is evident that the binding is primarily driven by van der Waals contribution. The van der Waals interactions were the predominant factor involved in protein-ligand interactions 84–86. Intercalative binding mode of Luteolin with DNA. Our MD simulation studies support the experimental evidences that luteolin binds to the DNA minor groove. Intercalation and groove binding are the two possible binding mechanisms for neutral molecules like luteolin. Molecular docking has a biasing nature to the initial model used for docking the drug and the initial receptor DNAs we have used for docking didn’t have an intercalation site. Although our biophysical studies indicate minor groove binding, the planar fused ring structure of luteolin suggests that luteolin could have a possible intercalative binding mode with DNA. There were procedures well reported on the generation of intercalation sites on a DNA sequence

87, 88

. To


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understand the intercalative binding mode of luteolin with DNA we have generated intercalation sites in DNA sequences (Figure S7) and have performed docking and further MD simulation studies on the DNA–luteolin intercalation complexes. The detailed discussion is given in the Supporting Information. The 20 ns unrestrained MD simulations of the two intercalation complexes dneAT_lut and cgcGC_lut were stable as evident from the rms deviations (Figure S8a–c). The binding free energies obtained were -13.91 kcal mol-1 and -12.89 kcal mol-1 for dneAT_lut and cgcGC_lut complexes respectively. There is no much significant preference is observed for GC or AT intercalation sites. Thus intercalation could also be a possible binding mode of action for luteolin molecule although our experimental results do not support the same. CONCLUSIONS. Flavonoids are bioactive compounds generally found in plants, exhibiting the potential to intervene in the physiological activities of our body. In this work, interaction of the flavanoid luteolin with DNA was explored through ITC, UV-Vis absorption, Melting temperature analysis, Differential Scanning Calorimetry, and MD simulation methods. ITC result shows that the binding reaction of luteolin with ctDNA is exothermic and enthalpy driven but costing in entropic part. Four binding sites were obtained and the binding constant of luteolin with ctDNA is in the range 106 to 104 M-1. Favorable negative enthalpies of binding and unfavorable entropic contributions are deduced from ITC. The effect of temperature and ion concentration on the binding of luteolin to ctDNA was analysed with UV–Vis spectroscopic titration experiments. The hypochromic effect without significant change in λmax is a clear evidence for the groove binding of luteolin. The decrease in binding constant on increasing Na+ ion concentration reveals a groove binding of luteolin with ctDNA. An electrostatic contribution could be inferred which is obvious from the hydroxyl groups of luteolin. The insignificant change in melting temperature of ctDNA upon luteolin binding as observed with the


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concentration dependent measurement of the value of Tm confirms a non-intercalative binding of luteolin with ctDNA which is also supported by DSC results. To have a better understanding of the interaction of luteolin with DNA, we have performed MD simulations on AT and GC rich DNA sequences in complex with luteolin. Conformational and free energy analysis of the DNA–luteolin systems show that the association of luteolin with the DNA duplexes is stable throughout the simulation period. The starting B form geometry of the DNA duplexes is retained with only slight variations in the average helical parameters and the sugar pucker. There is a reduction in the minor groove width around the ligand bound region which plays a major role in the stabilization of the complex as found in DNA ligand complexes by X-ray diffraction. The interaction of luteolin with DNA is mainly favored by electrostatic and van der Waals forces. Stacking interaction of the ring systems in luteolin with DNA strands and Hydrogen bonding, both direct and water mediated supports the stable binding of luteolin with DNA. Weak as well as strong hydrogen bonds such as O–HO, O–HN, N–HO, C–HO, and C–HN are observed between luteolin and DNA duplexes. Luteolin displaces the water network in the minor groove and forms water mediated hydrogen bonds with DNA atoms which also contribute to the stable binding. Binding free energy analysis through MMPBSA further supports the stable binding of luteolin to DNA duplexes. The total binding energy obtained for at6lut is -6.93 kcal mol-1, for at4lut it is -5.58 kcal mol-1 and cgclut has the value -5.61 kcal mol-1, including the unfavorable entropic contribution. The binding free energy values obtained from MMPBSA are in agreement with the experimental binding free energy derived from ITC. Luteolin is a potential groove binder for both AT and GC rich DNA sequences. This must be due to the more planar nature of the ring systems in luteolin which drives the strands of the GC rich duplex to a more confined conformation. The intercalation binding mode of luteolin to DNA was


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also probed through docking and MD simulation studies which suggest that intercalation could also be a potential binding mode for the action of luteolin. However our biophysical studies suggest that luteolin binds to the minor groove of ctDNA under the present experimental conditions and is supported by computational results.

AUTHOR INFORMATION Corresponding Author.

Email*: [email protected], Phone: +91 9447141561, Fax: 0481

2730423

Notes. The authors declare no competing financial interest.

ACKNOWLEDGEMENTS Authors Mary Varughese, Haris P, and Aparna P express their gratitude towards the University Grants Commission for the Research Fellowship in Sciences for Meritorious Students. Authors are thankful to Prof. M. Haridas, Inter University Centre for Bioscience, Kannur University, Kerala India for the ITC instrumentation support.

ASSOCIATED CONTENT Supporting Information Available: Figure S1, Structure of luteolin molecule. Atom names, atom types (gaff) and partial atomic charges of the atoms are given. Figure S2, absorbance versus luteolin (LUT) concentration plots for the titration data corresponding to Figure 3. Figure S3, absorbance versus ctDNA concentration plots for the titration data corresponding to Figure 4. Figure S4, UV–Vis absorption spectra of luteolin on increasing ctDNA concentration at different salt concentration. Figure S5, absorbance versus ctDNA concentration plots for the titration data corresponding to Figure S4. Figure S6, Temperature-dependent absorption of luteolin alone at


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352 nm, Simulation studies on the intercalating binding mode of Luteolin with DNA. This material is available free of charge via the Internet at http://pubs.acs.org.

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