Energetics, Thermodynamics, and Molecular Recognition of

Article pubs.acs.org/jcim

Energetics, Thermodynamics, and Molecular Recognition of Piperine with DNA P. Haris,†,⊥ Varughese Mary,†,⊥ M. Haridas,§ and C. Sudarsanakumar*,†,‡ †

School of Pure and Applied Physics and ‡Center for High Performance Computing, Mahatma Gandhi University, Kottayam, Kerala 686560, India § Inter University Centre for Bioscience, Kannur University, Thalassery Campus, Palayad, Kerala 670661, India S Supporting Information *

ABSTRACT: Piperine, the bioactive phytochemical from black pepper (Piper nigrum L.), is a nontoxic natural compound exhibiting many physiological and pharmacological properties. They include antioxidant, anti-inflammatory, antimutagenic, antitumor, antiapoptotic, antigenotoxic, antiarthritic, antifungal, antimicrobial, antidepressant, anti-HBV, and gastro-protective activities. It also enhances the bioavailability of phytochemicals and drugs. The molecular mechanism of action of piperine with DNA has not yet been addressed, while its pharmacological activities have been reported. In this work we report for the first time the interaction of piperine molecule with DNA duplex. We have carried out UV−vis absorption and fluorescence spectroscopy to confirm the binding of piperine with calf thymus DNA (ctDNA). The energetics of interaction of piperine with ctDNA was monitored by isothermal titration calorimetry (ITC). Differential scanning calorimetry (DSC) and melting temperature (Tm) analysis were also performed, confirming a minor groove mode of binding of piperine with ctDNA. The binding free energy ΔG values obtained from molecular dynamics simulation studies agree well with ITC values and reveal a sequence dependent minor groove binding exhibiting a specificity toward AT rich sequences.

■

antiepilepsirine have antidepressant-like activities11 and are found to be mediated in part through the inhibition of Monoamine oxidase (MAO) activity.18,19 Piperine is a potential candidate for developing drugs for cutaneous leishmaniasis.20 The health promoting potentials of the piperine makes it a possible candidate for drugs targeting biomacromolecules. Nucleic acids are common targets for anticancer, antiviral, antifungal, and antibiotic drugs. Structural studies can provide information about the interactions between the DNA and the ligand. This can also provide the information about the threedimensional shape of the complex and the exact mode of binding whether it is intercalation or groove binding. For a clear understanding of the binding process the nature of the molecular forces governing the binding and the energetic contributions from specific molecular interactions need to be evaluated. The molecular mechanism of action of piperine with biomacromolecules is not fully explored. Adequate reports on the actions of piperine with macromolecules are not yet available in the literature. The UV irradiation and protein binding of piperine were reported.21 The binding of piperine with human serum albumin by hydrophobic interactions was explored using steady state and fluorescence spectroscopy.22 The interaction of piperine with bovine β-lactoglobulin was studied by circular dichroism (CD) spectroscopy.23 CD and

INTRODUCTION Piperine, the major bioactive phytochemical from black pepper (Piper nigrum L.), native to India has a great role in medicine and in food as a spice since ancient times. Black pepper is used in many ayurvedic medicines. Foreigners came to India especially to Kerala in search of black pepper, and it has much importance in the history of India. Black pepper is wellknown as the “King of Spices” and “Black Gold” due to its importance in food, health, culture, and commerce. Piperine has many pharmacological properties. There are many reports supporting the medicinal properties of piperine. They include antioxidant, anti-inflammatory,1,2 antimutagenic,3 antitumor,4 antiapoptotic,5 antigenotoxic,6,7 antiarthritic,8,9 antifungal,10 antimicrobial, antidepressant,11 anti-HBV,12 and gastro-protective activities.13 Piperine is known for its anti-inflammatory effect against adjuvant-induced arthritis attributed by suppression of inflammation and cartilage destruction.2 Piperine improves gastrointestinal functionality and is found to boost nutrient absorption.13 Piperine enhances the bioavailability of phytochemicals and drugs.14,15 Piperine enhances the bioavailability of curcumin as a substrate to the toxic drug effluent protein P-gp and could be a plausible lead molecule for the development of nontoxic P-gp inhibitors.14 The bioavailability of resveratol could increase if combined with piperine.15 Piperine controls the progression of tumor growth13,16 and assists in cognitive brain function defending the body from depression and Alzheimer’s.17 Piperine and its derivative © XXXX American Chemical Society

Received: August 18, 2015

A

DOI: 10.1021/acs.jcim.5b00514 J. Chem. Inf. Model. XXXX, XXX, XXX−XXX

Article

Journal of Chemical Information and Modeling

Isothermal Titration Calorimetry. The thermodynamic parameters associated with the interaction of the drug piperine to ctDNA were examined using Isothermal Titration Calorimetry (ITC). Approximately 0.05 mM ctDNA was prepared in phosphate buffer solution at pH 7.0 containing 100 mM NaCl. The ligand piperine at concentration 0.05 mM was prepared in the same buffer containing 2% chloroform. Prior to loading the samples to the ITC machine both ctDNA and piperine solutions were degassed. The calorimetric titrations were performed at the temperature 25 °C using VP-ITC isothermal titration calorimeter from Microcal (Northampton, MA, USA) as described in the manufacturer’s instruction manual. Initially 2 μL of the piperine solution was injected to sample cell from the rotating syringe. The volume of the other injections was kept as 10 μL. Ten seconds were taken for each injection and a time interval of 240 s was set between two consecutive injections to allow the endothermic peak resulting from the reaction to return to the baseline. A total of 28 injections were made. The reference power was set as 15 μcal, and the stirring speed was adjusted to 307 rpm. The enthalpy change between ctDNA and 2% chloroform solution is subtracted from the original value and the final data at the end of the injections was fitted by nonlinear least-square method using ORIGIN software from Microcal. The data were best fitted to a single site of binding model to get the binding constant (K), enthalpy change (ΔH), entropy change (ΔS), and number of binding sites (n). The binding free energy (ΔG) and the entropic contribution (TΔS) were calculated from the standard equations

absorption spectroscopy reveal that piperine interacts with chicken α1-acid glycoprotein.24 The binding or interaction of piperine with DNA is not yet reported. The structure of piperine is shown in Figure 1 (and Figure S1). In the present

Figure 1. Structure of piperine.

work we discuss the experimental evaluation of the binding interaction of piperine with calf thymus DNA as well as the computational analysis of the dynamics of piperine DNA interaction. Docking and molecular dynamics (MD) simulation methods can be used to evaluate the dynamics and stability of drug binding to DNA and for calculating the binding energy of the complexes. In order to explore the sequence selectivity of the binding of piperine to DNA, we have performed three unrestrained MD simulations of DNA−piperine complexes; piperine complexed with AT rich sequences d(CGCAAATTTGCG)2 and d(CGCGATATCGCG)2 and a GC rich sequence d(CCGGCGCCGG)2 designated as the at6pip, at4pip, and cgcpip systems, respectively.

■

MATERIALS AND METHODS Materials. Highly polymerized type I calf thymus DNA sodium salt, piperine and phosphate buffer solution (PBS) of pH 7 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 sodium salt was dissolved in phosphate buffer solution (PBS) of pH 7.0 and kept for 24 h at 4 °C with occasional stirring to dissolve to a clear solution and marked as stock solution. A small amount of ctDNA was dissolved in PBS and measured the intensity of absorbance maxima at A260 and A280 to check the concentration and purity of ctDNA. The A260/A280 ratios were determined as 1.8, confirming that ctDNA samples are free from protein sufficiently.25 UV−vis Absorption Spectroscopy. The absorption spectra were recorded on a Shimadzu UV-2500 double beam spectrophotometer equipped with a temperature controller. Observations were taken at temperatures 25, 35, and 45 °C with drug concentration varied from 0 to 1.9837 × 10−4 M and a constant ctDNA concentration of 1.0 × 10−4 M. The binding constant Kp was calculated from the ratio of the intercept to the slope of the 1/(A − Ao) versus 1/Cdrug graph. Here Ao and A are the absorbance of ctDNA at 260 nm in the absence and presence of the drug piperine. Fluorescence Spectroscopy. Fluorescence measurements were carried out using HORIBA Fluoromax Spectrofluorometer with a fluorescence cuvette of 1.0 cm path length. The slit width was set to 5 nm for excitation and 10 nm for emission beams. Fluorescence titrations were carried out by adding increasing amount of ctDNA to the cell containing constant concentration of piperine (50 μM). Emission spectra were recorded in the range of 400−650 nm at an excitation wavelength of 342 nm.

ΔG = −RT ln K

(1)

ΔG = ΔH − T ΔS

(2)

DNA Melting Technique. The DNA melting temperature experiments were carried out on a Shimadzu UV-2500 double beam spectrophotometer equipped with a temperature controller and monitored the absorbance intensities of the ctDNA at 260 nm in the absence and presence of the piperine at various temperatures. The temperature of the sample was maintained with a thermocouple attached to the sample holder. The absorbance intensities of the ctDNA and ctDNA−piperine complex at 260 nm were plotted as a function of temperature from 20 to 100 °C. The melting temperature (Tm) of ctDNA was determined as the transition midpoint. Differential Scanning Calorimetry. The differential scanning calorimetry (DSC) experiments were carried out in a Nano DSC (TA, Waters LLC, New Castle, USA). The reference and the sample solutions were previously degassed in a degassing station (TA, Waters LLC, New Castle, USA) so that bubbles formation upon heating is reduced to a minimum. Both the cells were loaded with the buffer solution, equilibrated at 20 °C for 15 min and scanned at 3 atm pressure from 20 to 110 °C at a scan rate of 1 °C min−1. The DSC thermograms of excess heat capacity versus temperature were analyzed using the NanoAnalyze software. Docking and Molecular Dynamics Simulation. Computational Methods. The piperine structure was taken from the Cambridge Crystallographic data center26 (CCDC ID: PIPINE10)27 and the starting receptor DNAs were taken from the protein data bank (PDB).28 The PDB IDs of the DNA structures taken for MD simulation studies are 2DND [d(CGCAAATTTGCG)2]29 with a distamycin molecule bound in the minor groove, 1DNE [dB


Article


Figure 2. UV−vis absorption spectra of ctDNA (100 μM) in the absence and presence of piperine (0−180.45 μM) in phosphate buffer solution (pH 7.0, 100 mM NaCl) at (a) 25, (b) 35, and (c) 45 °C. In the inset plot of 1/(A − A0) versus 1/drug concentration for piperine and ctDNA complexes, where A0 is the initial absorbance of ctDNA (260 nm) and A is the absorbance at different piperine concentrations with constant ctDNA concentration of 100 μM at pH 7.0.

PTRAJ. The trajectory was visualized and figures were generated using VMD.46 Helical parameters of the representative structures were calculated using 3DNA.47 The graphs were plotted with the plotting program XMGRACE. Free energy calculations were performed with the MMPBSA48−50 method as implemented in AMBER with single trajectory approach: only the simulation of the complex was performed and the snapshots of the complex, receptor as well as the ligand were extracted from this trajectory. The snapshots for the free energy analysis were taken from the 3 ns of the stable part of the 15 ns production run with an interval of 50 ps which provided a total of 60 frames. The binding free energy of a receptor−ligand system can be represented as51

(CGCGATATCGCG)2]30 with a netropsin molecule as the minor groove binder, and 1CGC [d(CCGGCGCCGG)2]31 for at6pip, at4pip, and cgcpip systems, respectively. The associated ligands and crystal waters were removed from the respective nucleic acid structures and minimized prior to docking with piperine molecule. CHIMERA32 was used for the preparation of the receptor DNA as well as the ligand mol2 files and DOCK6.633 was used for docking. The flexible docking protocol was used and the best scored binding mode was further refined using amberscore method34 to yield a better binding conformation. The nucleic acid−piperine complexes obtained from docking were used as initial complex conformations in MD simulations. MD simulations were done using sander module of AMBER1235 suite of programs. The piperine was first optimized at the HF/6-31G* level using the Gaussian 09 package36 and electrostatic potentials (ESP) were then generated using Merz−Kollman population analysis method.37 The partial atomic charges used for the molecular mechanics calculations were derived from the ESP using RESP38 program implemented in AMBER in consistent with the general AMBER force field (gaff).39 The parameters for nucleic acids were available in the AMBER database and the Cornell40 force field with ff99bsc041 parameters were used for nucleic acids in the current simulations. The same simulation protocol was used for all the three systems. The systems were built with xLEaP, Na+ counterions were used to neutralize and then solvated in a truncated octahedron box of TIP3P water.42 The waterbox extends to at least 11 Å from the solute in all directions. The simulation was performed with periodic boundary conditions and the electrostatic interactions were evaluated with particle mesh Ewald method.43,44 The solvent and the counterions were minimized first with restraints on the DNA and piperine and then the whole system was minimized. The system was then heated up to 300 K in 100 ps, with 10 kcal mol−1 Å−2 restraints on the solute at constant volume with temperature controlling using Langevin thermostat and then further equilibrated at constant pressure to equilibrate the density. The restraints were gradually decreased from 10 kcal mol−1 Å−2 to 5, 2.5, 1, 0.5, 0.1, and finally to 0.01 kcal mol−1 Å−2 with 50 ps of simulation at each value of the restraint. After the 400 ps of equilibration run, a 15 ns production simulation was carried out and atomic coordinates were saved at an interval of 1 ps. The conformations generated were clustered using MMTSB45 and the representative structure having least rms deviation from the centroid of the largest cluster was used for helical parameter calculations and comparisons. The rms deviation and the structural parameters were calculated using

ΔG bind = Gcomplex − [Greceptor + G ligand]

(3)

where ΔG = ⟨EMM⟩ + ⟨Gpolar‐solvation⟩ + ⟨Gnonpolar‐solvation⟩ − TS (4)

The molecular mechanical energy ⟨EMM⟩ = ⟨E internal⟩ + ⟨Eelectrostatic⟩ + ⟨EvdW ⟩

(5)

is calculated using the force-field equation, with SANDER using continuum solvent method without any cutoff. The first term, internal energy, is constituted by the bond, angle, and dihedral terms. Since we are using single trajectory approach, the difference in the term ΔEinternal is zero. The ΔEMM contains only electrostatic and van der Waals components. ⟨Gpolar‑solvation⟩ and ⟨Gnonpolar‑solvation⟩ are polar solvation and nonpolar solvation energies, respectively. The polar solvation component is calculated by solving the PB (Poisson− Boltzmann) equation which was done with PBSA,52 with internal dielectric constant of 1 and external dielectric constant of 80, with a grid spacing of 0.33 Å. Nonpolar solvation energies were calculated with the equation ΔGnonpolar = γA + β

(6)

Here γ and β are constants and were derived experimentally53 as γ = 0.00542 kcal mol−1 Å−2 and β = 0.92 kcal mol−1 and the solvent accessible surface area A was estimated using molsurf with a probe radius of 1.4 Å. S is the solute entropy and was estimated using nmode. The binding energy can then be represented as ΔG bind = ΔEMM + ΔGsolv − T ΔS C

(7)


Article


■

RESULTS AND DISCUSSION UV−vis Absorption Spectroscopy. The binding mode and affinity of DNA with drugs can be estimated from electronic absorption spectroscopy.54−56 To explore the binding mode and understand the temperature dependence of the binding constant, UV−vis absorption titration experiments were carried out at 25, 35, and 45 °C keeping the ctDNA concentration constant and the piperine concentration increased by adding equal amount of aliquots of drug. Figure 2 shows an increase in the intensity of the ctDNA band at 260 nm which is due to the interaction of piperine with DNA bases. The absorption peak of piperine at 342 nm shows a reduction in broadness without significant change in absorption maxima. There is no noticeable shift in the absorption maxima of piperine indicating that the structural alteration of the drug upon its complexation with ctDNA is minimal leading to the possibility of groove binding.57 To evaluate the binding constant of piperine and ctDNA interaction we used the procedure proposed by Stephanos and Zhong.58−60 The interaction reaction can be written as follows calf thymus DNA + piperine ⇔ ctDNA:piperine

Kp =

[ctDNA:piperine] [ctDNA][piperine]

Kp =

C PD (C D − C PD)(C P − C PD)

Table 1. Binding Constant of Interaction of Piperine with ctDNA at Different Temperatures from UV−vis Absorption Spectroscopy Titration Experiments

C PD =

A0 εD S (A − A 0 ) (εPD S)

R2

KP (M−1)

1 2 3

25 35 45

0.996 0.997 0.987

1.960 × 103 3.501 × 103 1.986 × 103

(8)

(9) Figure 3. UV−vis absorption spectra of ctDNA (100 μM) in the absence and presence of piperine (115.38 μM) in phosphate buffer solution pH 7.0 containing 100 mM NaCl. (a) ctDNA in the absence of piperine at 25, 35, and 45 °C and ctDNA in the presence of piperine at (b) 25, (c) 35, and (d) 45 °C.

(10)

the absorbance of ctDNA (100 μM) at 260 nm does not show any significant change upon increasing the temperature. However, in the presence of piperine at constant concentration of ctDNA (100 μM) and piperine (115.38 μM), the absorbance increases with increasing temperature for the absorption peak at 260 nm as well as 342 nm. Fluorescence Quenching Study. Piperine is a strong fluorophore that has a broad emission spectrum in the range of 400−650 nm with a band maximum at 486 nm on excitation with wavelength of 342 nm (Figure 4). Upon adding equal amount of aliquots of ctDNA with a fixed concentration of piperine, the fluorescence intensity of the drug piperine is

(11)

(12)

where A0 and A are the absorbance of ctDNA at 260 nm in the absence and presence of piperine, respectively. εD and εPD are the molar extinction coefficient of free ctDNA and piperine bound ctDNA respectively. S = 1 cm is the light path length of the cuvette. By substituting the values of CD and CPD in eq 10 from eqs 11 and 12, we will get A0 ε ε 1 = D + D A − A0 εPD εPDK C P

T (°C)

aminobenzothiazole with herring sperm DNA. The binding constant of curcumin with ctDNA was reported by Nafisi et al. as 4.25 × 104 M−1.61 Figure 3 shows the temperature dependence of the interaction of piperine with ctDNA. In the absence of piperine,

where KP is the binding constant, CPD is the concentration of ctDNA and piperine complex, CD is the concentration of ctDNA, and CP is the concentration of the drug piperine, respectively. To find out the concentration of DNA we used the Beer−Lambert law CD =

Sl no:

(13)

The binding constant of the interaction of piperine with ctDNA (KP) is the ratio of the intercept to the slope of the linear double reciprocal graph of A0/(A − A0) vs 1/CP. The values of the binding constant obtained at different temperatures are tabulated in Table 1. The binding constant at 35 °C (3.501 × 103 M−1) is more than the corresponding values at 25 °C (1.960 × 103 M−1) and 45 °C (1.986 × 103 M−1) with regression coefficients 0.997, 0.996, and 0.987, respectively. This result indicates that in a body temperature environment, piperine may interact more efficiently with DNA. This highest value at 35 °C is comparable to the values reported for some minor groove binding drugs.60,61 A binding constant of 7.2 × 103 M−1 60 have been reported for the minor groove binder 2-

Figure 4. (a) Fluorescence emission spectra of piperine (50 × 10−5 M) in the presence of increasing concentrations of ctDNA (0−45.33 × 10−6 M) on excitation at 342 nm. (b) Stern−Volmer plot. (c) Log[(F0 − F)/F] versus log[ctDNA] plot of the fluorescence quenching of piperine with different concentrations of ctDNA. D


Article


to a single injection of piperine into ctDNA solution. The thermogram for the binding of piperine to ctDNA was obtained by integrating the area under each peak and subtracting the heats of dilution. The lower panel of Figure 5 was obtained by plotting the corrected heat as a function of molar ratio of piperine and ctDNA. The experimental injection heats were represented by solid rectangular points and the calculated fit of the data represented by a solid line. From the ITC profile it is clear that the binding was exothermic in each injection and there was only a single binding mode. The data were best fitted to a single site of binding model. The binding affinity of piperine to ctDNA (Kb) is 5.95 × 104 M−1, and the number of binding sites is 0.629. The enthalpy of binding (ΔH) is −11.030 kcal mol−1, the negative sign reflects that the reaction was exothermic and enthalpy driven. The entropy of binding reaction (−TΔS) and Gibb’s free energy (ΔG) were determined from the eqs 1 and 2 as +4.502 and −6.528 kcal mol−1, respectively. The binding parameters obtained are presented in Table 2. Similar values of binding parameters for the binding of anti cancer drug curcumin in the minor groove of DNA were reported63 where Kb = 4.03 × 104 M−1, ΔH = −4.355 kcal mol−1, TΔS = 1.927 kcal mol−1, and ΔG = −6.282 kcal mol−1 at T = 25 °C. The thermodynamic parameters of the drug piperine and ctDNA are also close to the corresponding reported values of minor groove binders distamycin and netropsin.64 Another minor groove binder NPOS was reported to bind with ctDNA with a favorable negative enthalpy and Gibbs free energy like that we obtained for piperine and ctDNA.57 The minor groove binder Thiazotropsin A binding in 2:1 fashion was also reported to have similar negative enthalpy, Gibbs free energy as well as an unfavorable entropy contribution.65 Thermodynamic parameters for the interaction of piperine with ctDNA reveal that piperine binds on the minor groove of the DNA and that the binding process is enthalpy driven associated with an entropic penalty. Melting Temperature Analysis. Melting temperature analysis is an important tool to evaluate the mode of binding of the drug with DNA. The melting temperature of DNA may change on interaction with small molecules. An increase in melting temperature of about 5−8 °C is reported to occur if the drug intercalate to the double helix of the DNA resulting in an increase in the stability of the structure of DNA, while in the case of non intercalating interaction, not much increase in melting temperature (Tm) is observed.66−68 The melting temperature curves of ctDNA in the absence and presence of piperine are shown in Figure 6. The melting temperature (Tm) of ctDNA alone is 86.7 °C and that of ctDNA−piperine complex is 87.3 °C. The smaller change in melting temperature, Tm of ctDNA observed in the presence of piperine indicates the minor groove binding of piperine to ctDNA. Similar variation in the melting temperature was reported for the minor groove binding of quercetin, kaempferide, and luteolin with DNA.68 The melting temperature analysis suggests that piperine binds into the minor groove of ctDNA. Differential Scanning Calorimetry. A huge amount of thermodynamic information on the drug DNA interaction may be obtained from a single DSC experiment. The thermodynamic parameters obtained from DSC are given in Table 3. The enhancement in melting temperature of ctDNA upon piperine binding as obtained from DSC is 0.5 °C which is in agreement with that from the melting analysis. The specific heat capacity,

gradually quenched which can be analyzed by the Stern− Volmer equation. F0/F = 1 + Kqτ0[Q ] = 1 + K sv[Q ]

(14)

where F0 and F are the fluorescence intensities of piperine in the absence and presence of ctDNA. Kq is the quenching rate constant, Ksv is the Stern−Volmer quenching constant, [Q] is the concentration of ctDNA, and τ0 is the average lifetime of biomolecule without quencher. The default value of τ0 is 10−8 s.62 The Stern−Volmer quenching constant Ksv determined from the slope of the plot of F0/F against [ctDNA] is 2.6 × 103 M−1, and the quenching rate constant Kq is then calculated to be 2.6 × 1011 M−1 s−1. The binding constant Kp and the number of binding sites n of piperine with ctDNA can be calculated from the following equation: log[(F0 − F )/F ] = log K p + n log[Q ]

(15)

From the linear plot of log(F0 − F)/F versus log[ctDNA], the values Kp and n are obtained as 1.25 × 103 M−1 and 0.855 respectively. In this study, the binding constant obtained from fluorescence spectroscopic titration and absorption spectroscopy are in agreement with each other. Isothermal Titration Calorimetry. Isothermal titration calorimetry (ITC) is an important tool to investigate the thermodynamic parameters of drug−DNA interaction. From a single ITC experiment, we will get a complete thermodynamic profile of drug−DNA interaction along with the number of binding sites and binding constant. The ITC profile of the binding of piperine with ctDNA is shown in Figure 5. The upper panel of the figure represents the raw ITC curves resulting from the injection of piperine to the solution of ctDNA. Each of the heat burst curves in the figure corresponds

Figure 5. ITC data for the titration of piperine into ctDNA (PBS buffer, pH 7.0) at 25 °C. The top panel represents the heat burst curves for successive injection of aliquots of piperine into ctDNA. The bottom panel represents the corresponding normalized heat signals versus molar ratio fitted to one site model. E


Article

Journal of Chemical Information and Modeling Table 2. Binding Parameters of Piperine to ctDNA ligand

K

T

−TΔS

ΔH

ΔG

M−1

K

kcal mol−1

kcal mol−1

kcal mol−1

5.95 × 104

298.15

+4.502

−11.030

−6.528

receptor

piperine

ctDNA

Figure 6. Thermal melting profiles of ctDNA (10 × 10−6 M) in the (a) absence and (b) presence of piperine (20 × 10−6 M) at 260 nm.

Figure 7. DSC curves for thermal melting of ctDNA (3.0 × 10−4 M) in the presence and absence of piperine (9.0 × 10−5 M).

enthalpy, entropy, and Gibbs free energy values obtained from DSC at the melting temperatures in the absence and presence of piperine does not show any dramatic changes. Figure 7 shows the DSC curves for thermal melting of ctDNA (3.0 × 10−4 M) in the presence and absence of piperine (9.0 × 10−5 M). This establishes, in support to ITC and melting analysis results, that piperine is a minor groove binder to DNA. The negative change in ΔG by ∼3.2 kcal mol−1 indicates a stable binding of piperine with ctDNA. Molecular Dynamics Simulation Study. The 15 ns unrestrained MD simulations of the three complexes at6pip, at4pip, and cgcpip were stable as indicated by the rms deviations (Figure 8). The piperine was bound to the duplex throughout the simulations. The rms deviation of the at6pip duplex structure is stabilized at an average value of ∼1.75 Å and that of piperine is 0.5 Å, low as expected. The average rms deviation of the at4pip structure is ∼2 Å while that of piperine is ∼0.6 Å. In the cgcpip system the rms deviation is ∼2.4 Å and that of piperine is ∼0.65 Å. The rms deviations of the three complexes show that the binding of piperine with at6 duplex is more stable compared to at4 and cgc duplexes. The potential and kinetic energies of the trajectories were also stabilized after the equilibration run. In the at6pip system, all the base pair hydrogen bonds in the duplex were retained throughout the simulation with high occupancies. Of the 30 expected base pair hydrogen bonds, 24 were having occupancies greater than 98%, 4 show occupancies greater than 85%, and the remaining 2 terminal hydrogen bonds show occupancies greater than 50%. In the at4pip system, 23 base pair hydrogen bonds were observed with occupancies greater than 98%, 6 were found with occupancies greater than 85%, and the remaining 3 terminal hydrogen bonds have occupancies greater than 70%. In the cgcpip system, among the 30 expected base-pair hydrogen bonds 27 have occupancies greater than 98% and the remaining 3 have occupancies greater than 90%. Thus, in all the systems base pair

Figure 8. Backbone rms deviations of DNA (black) and piperine (red) from the starting structures in different trajectories.

hydrogen bonds were very much retained showing that the duplex systems are intact throughout the simulations. DNA Conformation and Drug Binding Interactions. The conformations generated in the simulations were clustered using MMTSB. The structure with lowest rms deviation from the centroid of each cluster was taken as the representative structure and used for the calculation of helical parameters. The number of conformations in different clusters and rms deviations of the representative structures from the corresponding starting structures is given in Table S1. In at6pip system, two clusters were obtained with 8532 conformations in the first and 6468 conformations in the second. The rms deviation between the representative structures of clusters1 and 2 is 1.18 Å for the duplex. The corresponding rms deviation of piperine molecule (0.3 Å) is low as expected. Figure 9a and b shows the starting structure and the superposition of representative structures from each cluster of at6pip. In the most populated cluster of at6pip, cluster1, the piperine molecule is bound

Table 3. Thermodynamic parameters of ctDNA and the ctDNA−Piperine System system ctDNA (300 μM) ctDNA (300 μM) + piperine (90 μM)

Tm

dCp

dH

ΔH

ΔS

ΔG

°C

kcal mol−1 K−1

kcal mol−1

kcal mol−1

kcal mol−1 K−1

kcal mol−1

87.5 88.0

4.766 4.799

359.37 409.07

902.084 845.281

2.4956 2.3438

2.046 −1.182

F


Article


Figure 9. (a) Starting structure. (b) Superposition of the representative structures of the two clusters in at6pip (cluster1 blue, cluster2 red). (c) Hydrogen bonds observed in cluster1.

complex has a reduced minor groove width of 5.5 Å in the binding region for the most populated cluster. The minor groove widths of AT rich DNA duplexes on complex with curcumin molecule are given in Table 4; values are quoted for the most populated clusters only. Similar minor groove width patterns were observed for other DNA minor groove complexes29,30,70−72 which are also tabulated in Table 4. Piperine interacts with the at6 duplex through hydrogen bonds. The C−H···O and C−H···N hydrogen bonds observed between piperine and duplex base as well as backbone atoms of the duplex are given in Table S2. Figure 9c is a schematic depiction of the hydrogen bonds observed in cluster1. In the second system, at4pip, the conformations generated were clustered into two clusters, with 13011 in cluster1 and 1989 in cluster2. Starting structure and the superposition of representative structures of the clusters are shown in Figure 10a,b. The rms deviation between cluster1 and 2 is 1.7 Å for the duplex and that for the piperine is 0.37 Å. In the most crowded cluster1, the piperine is bound between the region A6•T19:T7•A18:T8•A17:C9•G16:G10•C15, aligned in the center of the minor groove as in at6pip whereas in cluster2 the ligand has moved a base step toward 5′ end. The average rise, twist, and the number of residues per turn are 3.2 Å, 35.9°, and 10 respectively in cluster1. The minor groove width is 5.46 Å in the binding region and 8.00 Å at other regions; consistent with an at4−curcumin simulation where a reduced minor groove width of 5.4 Å at the binding site was observed.69 C−H···O, N−H···O, and C−H···N hydrogen bonds are observed between the ligand and duplex base as well as backbone atoms. Hydrogen bonds observed in the representative structure of both clusters are given in Table S2, and a schematic illustration of the hydrogen bonds observed in cluster1 is also given in Figure 10c. In the cgcpip system, 2 clusters were obtained with 9160 conformations in cluster1 and 5840 conformations in cluster2. The rms deviation between the representative structures of the two clusters is 1.63 Å for the duplex and the corresponding rms deviation of piperine is 0.88 Å. Figure 11a and b shows the starting structure and the superposition of representative structures of the clusters. In cluster1, the most populated one, piperine is bound between the region C2•G19:G3•C18:G4•C17:C5•G16:G6•C15, aligned closer

between the region A4•T21:A5•T20:A6•T19:T7•A18:T8•A17; the regions of close contact are shown in bold, arranged 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 3.1 Å and 34.7° and the number of base pairs per turn is 10.4. Table 4 shows the minor groove widths near and away from the Table 4. Minor Groove Widths of the Representative Structures of Different Clusters of the Three Systems at the Ligand Bound Region and the Region Away from the Ligand, Minor Groove Widths Reported from DNA− Curcumin Simulations, and Minor Groove Widths of Some Crystal Structures of Minor Groove Complexes minor groove width (Å) system cluster1 cluster2 cluster1 cluster2 cluster1 cluster2 at6-curcumin69 at4-curcumin69 PDB ID (ligand) 2DND (distamycin)29 1DNE (netropsin)30 121D (netropsin)70 1D63 (berenil)71 1LEX (lexitropsin)72

near the ligand at6pip 4.77 5.36 at4pip 5.46 5.9 cgcpip 7.77 7.87 5.5 5.4 Crystal Structures 4.62 4.49 4.57 4.82 4.68

away from the ligand 7.48 8.08 8.00 8.64 8.22 8.42 7.4 8.3

6.81 6.44 6.64 6.47 7.29

ligand binding region in different clusters from the three systems. The minor groove width is reduced to 4.77 Å at the binding region and 7.48 Å at other regions in cluster1. Such a reduction in minor groove width at the binding site was reported to be observed in the simulation study of DNA− curcumin complexes.69 It was observed that the at6−curcumin G


Article


Figure 10. (a) Starting structure. (b) Superposition of the representative structures of the two clusters in at4pip (cluster1 blue, cluster2 red). (c) Hydrogen bonds observed in cluster1.

Figure 11. (a) Starting structure. (b) Superposition of the representative structures of the two clusters in cgcpip (cluster1 blue, cluster2 red). (c) Hydrogen bonds observed in cluster1.

Figure 12. Water mediated contacts in (a) at6pip, (b) at4pip, and (c) cgcpip systems.

to the first strand whereas in cluster2 the ligand has moved a base step toward 5′ end and aligned closer to the second strand. The average rise and twist are 3.1 Å and 32.3° respectively and the number of residues per turn is 11.1 in cluster1. The minor groove widths are 7.77 and 8.22 Å near and away from the ligand, respectively. In cluster1, only one close contact is observed (Figure 11c and Table S2); an N−H···O hydrogen

bond of piperine:O3 with G3:N2 (2.97 Å) of cgc duplex. In cluster2, there are three hydrogen bonds (Table S2); piperine:O3 with G19:N2 (2.83 Å), C5 with C18:O4′ (3.25 Å), and C4 with C5:O4′ (3.28 Å). In all the three systems the backbone torsion angles were distributed in standard conformations. The α−γ torsions were in the preferred values except for some variations which might H


Article


Figure 13. Hydration pattern of the (a) at6pip, (b) at4pip, and (c) cgcpip systems. Water density is contoured at ∼2.5 times the bulk water density.

Table 5. Binding Energy Components of Three Complexesa

a

complex

ΔEELE

ΔEvdW

ΔEMM

ΔGpol

ΔGn‑pol

ΔGSOL

ΔPBTOT

ΔTSTOT

ΔG

at6pip at4pip cgcpip

−13.17 −10.42 −7.73

−45.08 −44.57 −38.94

−58.25 −54.99 −46.67

36.71 33.00 27.52

−4.67 −4.65 −4.34

32.04 28.35 23.18

−26.21 −26.65 −23.49

−18.46 −19.08 −20.31

−7.75 −6.52 −3.18

ΔEMM = ΔEELE + ΔEvdW, ΔGSOL = ΔGpol + ΔGn‑pol, ΔPBTOT = ΔEMM + ΔGSOL, ΔG = ΔPBTOT − ΔTSTOT. All values are in kilocalories per mole.

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 at6pip, at4pip, and cgcpip the sugar puckers were mainly distributed among C2′ endo and C1′ exo, both belong to the B-form family. O4′ endo sugar pucker which is the lowest energy intermediate between A and B forms were also seen for some residues; this was also reported in the starting crystal structures. Hydration and Water-Mediated Binding Interactions. Water molecules play an important role in the binding of ligands to the DNA duplexes. The representative structure of the largest cluster in each system is extracted and the water mediated contacts between piperine and the DNA duplex atoms were analyzed. The water network in the minor groove is found to be adjusted to incorporate the piperine molecule. Piperine exhibits water mediated interactions with DNA through two or more water molecules. Figure 12 shows the water mediated interactions observed between the piperine and DNA atoms in the three systems. In at6pip and cgcpip, piperine:O1 and O2 atoms are involved in the water network whereas in at4pip only the piperine:O1 atom is found to be participating in the network of water mediated interactions. The water mediated hydrogen bonds observed in the three systems ranges from 1.60 to 3.01 Å. Water molecules are reported to have crucial role in the DNA minor groove recognition by small molecules.73 Similar water mediated contacts between DNA and ligands were observed in MD simulations of DNA minor groove complexes with DAPI.69,74 Hydrations of the duplexes were examined with hydrogen bonding and grid analysis facilities of ptraj module. As expected, the phosphates and the minor groove of the duplexes were hydrated except for the piperine bound region. Figure 13 shows the hydration pattern of at6pip, at4pip, and cgcpip, respectively, obtained from the grid analysis contoured at ∼2.5 times the bulk water density. In all the three systems on an average ∼3.4 water molecules were in contact with the phosphates throughout the simulation.

There is a spine of hydration in the minor groove with variations in the region of the ligand. Water molecules are present near the O1 and O2 atoms of piperine showing high residence times in all the systems. The hydration pattern of the major groove is similar to that of normal BDNA with long residence times.75 Such a deviation in the hydration pattern of DNA duplexes is also observed in other minor groove complexes.69,74 Free Energy Calculations. The firm binding of the piperine molecule in the minor groove is confirmed by the structural stability of the DNA−piperine complexes. MM-PBSA free energy analysis on the MD simulation trajectories of the DNA− piperine complexes further supports this view quantitatively. The values of binding energy components calculated for at6pip, at4pip, and cgcpip systems are summarized in Table 5. In all the three systems, van der Waals and electrostatic contributions are favorable for the DNA−piperine binding. While the nonpolar component of the solvation free energy is favorable, the polar component is highly unfavorable leading to 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 complex formation.76 NMODE calculations overestimate the rotational/translational entropy of binding. However, the binding free energies between piperine and DNA duplexes which include the solute entropic contribution are appropriate for our work. The binding energy obtained for at6pip is −7.75 kcal mol−1, for at4pip it is −6.52 kcal mol−1 and cgcpip has the lowest value −3.18 kcal mol−1, including the unfavorable entropic contribution. In the case of at6pip, van der Waals, and molecular mechanical electrostatic interactions are more favorable than at4pip and cgcpip and hence the total binding free energy obtained is the highest among the three. Comparatively low van der Waals contribution is seen in cgcpip system which may be due to the wider minor groove. From the results it is evident that the DNA−piperine binding is primarily steered by van der Waals I


Article


hydrogen bonds with DNA atoms mediated through two or more water molecules. Such water mediated hydrogen bonds also contribute to the stability of DNA−piperine binding. The phosphates, minor groove, and major groove are hydrated in all the three systems. The piperine binding modifies the spine of hydration in the minor groove. MM-PBSA studies indicate that the noncovalent binding of the piperine in the minor grooves of the duplexes is stable and the binding free energies obtained are favorable for piperine binding. The total binding free energy calculated for at6pip is −7.75 kcal mol−1, for at4pip it is −6.52 kcal mol−1, and cgcpip has the lowest value −3.18 kcal mol−1. The values obtained are in agreement with the experimental binding free energy (−6.528 kcal mol−1) derived from ITC. The cgcpip system has the lowest van der Waals contribution among the three systems which may account for the wider minor groove. The binding energy values obtained as well as the variations in helical parameters may indicate the selectivity of the piperine molecule to AT rich sequences over the GC rich sequence which is in agreement with AT rich sequence selectivity characteristic of minor groove binders. Thus, in conclusion, a sequence dependent interaction of piperine with DNA in the minor groove binding could be confirmed by experimental and theoretical studies.

contribution. This is presumed as the molecule has some shape similarity with the minor groove of the duplex and is electrically neutral. The van der Waals interactions play a significant role in protein−ligand interactions also.50,76,77 The binding free energies between piperine and DNA duplexes obtained by MM-PBSA single trajectory approach were in agreement with the experimental results. Thus, the present work demonstrates that docking followed by MD simulations with AMBER are able to compliment the experimental DNA drug binding interactions. The MM-PBSA is an established method used in the study of a variety of systems containing nucleic acids.48,49,69,74 Though simple, the applied AMBER implemented MM-PBSA technique provides a fairly approximate calculation of binding energy based on continuum representation of the solvent, and this methodology is appropriate for our DNA−piperine system.

■

CONCLUSION In this work, we have explored the molecular mechanism of interaction between piperine and ctDNA through UV−vis absorption, fluorescence, ITC, DSC, and melting temperature analysis as well as MD simulation methods. UV−vis absorption and fluorescence spectroscopic studies indicate that piperine binds to ctDNA as evidenced by hypochromism and quenching constant. The energetics of interaction of piperine with ctDNA was monitored by ITC. The binding affinity of piperine to ctDNA is 5.95 × 104 M−1, the Gibb’s free energy is −6.528 kcal mol−1, and the number of binding sites is 0.629. A negative enthalpy of binding reflects that the reaction was exothermic and enthalpy driven. DSC and melting temperature analysis further confirmed the minor groove mode of binding of piperine with ctDNA. On the basis of experimental evidence as well as the shape similarity of the piperine molecule to other minor groove binders, we have performed MD simulations of some DNA− piperine complexes. The goal behind the simulation study is to have an understanding of the sequence selectivity of the piperine as a minor groove binder toward AT and GC rich nucleic acid sequences as well as the mode of binding and evaluate the binding energy of the complexes. We have docked piperine molecule to DNA duplexes and free energy analysis of the DNA−piperine complexes were performed to assess the binding. Our results show that the association of piperine with the duplexes is stable in all the three systems, though the GC rich sequence shows a significant reduction in stability which was reflected in the RMS deviation, helical parameters, hydrogen bonding, and the binding energy of the complex. The interaction of piperine with DNA is mainly favored by van der Waals forces, as presumed upon the charge neutrality and also on the hydrogen bonding, both direct and water mediated. The starting B form geometry of the DNA duplexes is retained in all three systems with only slight variations in the average helical parameters and the sugar pucker. However, the helical parameters show variations in the ligand bound region. The groove widths are reduced by about 2 Å in the piperine bound region in all of the systems and show higher values in other regions. This variation in groove width observed is an important factor in the stabilization of the complex. The binding of the piperine was stable in all the three systems and the interactions are mainly governed by van der Waals and hydrogen bonding (weak as well as strong) such as C−H···N, C−H···O, and N−H···O. The piperine molecule displaces the normal water network around the DNA duplexes and forms

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.5b00514. Figure S1, structure of piperine molecule with atom names and atom types (gaff) of heavy atoms only. Table S1, number of conformations in different clusters and rms deviations of the representative structures from the corresponding starting structures. Table S2, hydrogen bonds observed between the piperine atoms and the DNA duplex atoms in different clusters of the three simulations (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +91 9447141561. Fax: 0481 2730423. Author Contributions ⊥

The first and second authors have contributed equally to this work.

Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Authors P.H. and V.M. are thankful to the University Grants Commission for the Research Fellowship in Sciences for Meritorious Students. The authors thank Dr. S. Karthikeyan, Institute of Microbial Technology, Chandigarh, India, for help with DSC analysis.

■

REFERENCES

(1) Shrivastava, P.; Vaibhav, K.; Tabassum, R.; Khan, A.; Ishrat, T.; Khan, M. M.; Ahmad, A.; Islam, F.; Safhi, M. M.; Islam, F. Antiapoptotic and Anti-inflammatory Effect of Piperine on 6-OHDA Induced Parkinson’s Rat Model. J. Nutr. Biochem. 2013, 24, 680−687. (2) Murunikkara, V.; Pragasam, S. J.; Kodandaraman, G.; Sabina, E. P.; Rasool, M. Anti-inflammatory Effect of Piperine in Adjuvant-

J


Article

Journal of Chemical Information and Modeling Induced Arthritic Rats-a Biochemical Approach. Inflammation 2012, 35, 1348−1356. (3) Srinivasan, K. Black Pepper and its Pungent Principle-Piperine: A Review of Diverse Physiological Effects. Crit. Rev. Food Sci. Nutr. 2007, 47, 735−748. (4) Makhov, P.; Golovine, K.; Canter, D.; Kutikov, A.; Simhan, J.; Corlew, M. M.; Uzzo, R. G.; Kolenko, V. M. Co-Administration of Piperine and Docetaxel Results in Improved Anti-Tumor Efficacy via Inhibition of CYP3A4 activity. Prostate 2012, 72, 661−667. (5) Lee, W.; Kim, K. Y.; Yu, S. N.; Kim, S. H.; Chun, S. S.; Ji, J. H.; Yu, H. S.; Ahn, S. C. Pipernonaline from Piper longum Linn. Induces ROS-Mediated Apoptosis in Human Prostate Cancer PC-3 Cells. Biochem. Biophys. Res. Commun. 2013, 430, 406−412. (6) Doucette, C. D.; Hilchie, A. L.; Liwski, R.; Hoskin, D. W. Piperine, a Dietary Phytochemical, Inhibits Angiogenesis. J. Nutr. Biochem. 2013, 24, 231−239. (7) Balakrishnan, S.; Vellaichamy, L.; Menon, V. P.; Manoharan, S. Antigenotoxic Effects of Curcumin and Piperine Alone or in Combination Against 7,12-Dimethylbenz(a)anthracene Induced Genotoxicity in Bone Marrow of Golden Syrian Hamsters. Toxicol. Mech. Methods 2008, 18, 691−696. (8) Bang, J. S.; Oh, D. H.; Choi, H. M.; Sur, B. J.; Lim, S. J.; Kim, J. Y.; Yang, H. I.; Yoo, M. C.; Hahm, D. H.; Kim, K. S. Antiinflammatory and Antiarthritic Effects of Piperine in Human Interleukin 1β-Stimulated Fibroblast-Like Synoviocytes and in Rat Arthritis Models. Arthritis Res. Ther. 2009, 11, R49. (9) Umar, S.; Golam Sarwar, A. H.; Umar, K.; Ahmad, N.; Sajad, M.; Ahmad, S.; Katiyar, C. K.; Khan, H. A. Piperine Ameliorates Oxidative Stress, Inflammation and Histological Outcome in Collagen Induced Arthritis. Cell. Immunol. 2013, 284, 51−59. (10) Kumar, A.; Raman, R. P.; Kumar, K.; Pandey, P. K.; Kumar, V.; Mohanty, S.; Kumar, S. Antiparasitic Efficacy of Piperine Against Argulus spp. on Carassius auratus (Linn. 1758): in Vitro and in Vivo Study. Parasitol. Res. 2012, 111, 2071−2076. (11) Li, S.; Wang, C.; Li, W.; Koike, K.; Nikaido, T.; Wang, M. W. Antidepressant-Like Effects of Piperine and its Derivative, Antiepilepsirine. J. Asian Nat. Prod. Res. 2007, 9, 421−430. (12) Jiang, Z. Y.; Liu, W. F.; Zhang, X. M.; Luo, J.; Ma, Y. B.; Chen, J. J. Anti-HBV Active Constituents from Piper longum. Bioorg. Med. Chem. Lett. 2013, 23, 2123−2127. (13) Butt, M. S.; Pasha, I.; Sultan, M. T.; Randhawa, M. A.; Saeed, F.; Ahmed, W. Black Pepper and Health Claims: A Comprehensive Treatise. Crit. Rev. Food Sci. Nutr. 2013, 53, 875−886. (14) Singh, D. V.; Godbole, M. M.; Misra, K. A Plausible Explanation for Enhanced Bioavailability of P-gp Substrates in Presence of Piperine: Simulation for Next Generation of P-gp Inhibitors. J. Mol. Model. 2013, 19, 227−238. (15) Johnson, J. J.; Nihal, M.; Siddiqui, I. A.; Scarlett, C. O.; Bailey, H. H.; Mukhtar, H.; Ahmad, N. Enhancing the Bioavailability of Resveratrol by Combining it with Piperine. Mol. Nutr. Food Res. 2011, 55, 1169−1176. (16) Yaffe, P. B.; Doucette, C. D.; Walsh, M.; Hoskin, D. W. Piperine Impairs Cell Cycle Progression and Causes Reactive Oxygen SpeciesDependent Apoptosis in Rectal Cancer Cells. Exp. Mol. Pathol. 2013, 94, 109−114. (17) Parachikova, A.; Green, K. N.; Hendrix, C.; LaFerla, F. M. Formulation of a Medical Food Cocktail for Alzheimer’s Disease: Beneficial Effects on Cognition and Neuropathology in a Mouse Model of the Disease. PLoS One 2010, 5, e14015. (18) Lee, S. A.; Hong, S. S.; Han, X. H.; Hwang, J. S.; Oh, G. J.; Lee, K. S.; Lee, M. K.; Hwang, B. Y.; Ro, J. S. Piperine from the Fruits of Piper longum with Inhibitory Effect on Monoamine Oxidase and Antidepressant-Like Activity. Chem. Pharm. Bull. 2005, 53, 832−835. (19) Rahman, T.; Rahmatullah, M. Proposed Structural Basis of Interaction of Piperine and Related Compounds with Monoamine Oxidases. Bioorg. Med. Chem. Lett. 2010, 20, 537−540. (20) Ferreira, C.; Soares, D. C.; Barreto-Junior, C. B.; Nascimento, M. T.; Freire-de-Lima, L.; Delorenzi, J. C.; Lima, M. E. F.; Atella, G. C.; Folly, E.; Carvalho, T. M. U.; Saraiva, E. M.; Pinto-da-Silva, L. H.

Leishmanicidal Effects of Piperine, its Derivatives and Analogues on Leishmania amazonensis. Phytochemistry 2011, 72, 2155−2164. (21) Soumyanath, A.; Venkatasamy, R.; Joshi, M.; Faas, L.; Adejuyigbe, B.; Drake, A. F.; Hider, R. C.; Young, A. R. UV Irradiation Affects Melanocyte Stimulatory Activity and Protein Binding of Piperine. Photochem. Photobiol. 2006, 82, 1541−1548. (22) Suresh, D. V.; Mahesha, H. G.; Rao, A. G.; Srinivasan, K. Binding of Bioactive Phytochemical Piperine with Human Serum Albumin: A Spectrofluorometric Study. Biopolymers 2007, 86, 265− 275. (23) Zsila, F.; Hazai, E.; Sawyer, L. Binding of the Pepper Alkaloid Piperine to Bovine β-Lactoglobulin: Circular Dichroism Spectroscopy and Molecular Modeling Study. J. Agric. Food Chem. 2005, 53, 10179− 10185. (24) Zsila, F.; Matsunaga, H.; Bikadi, Z.; Haginaka, J. Multiple Ligand-Binding Properties of the Lipocalin Member Chicken α1-Acid Glycoprotein Studied by Circular Dichroism and Electronic Absorption Spectroscopy: The Essential Role of the Conserved Tryptophan Residue. Biochim. Biophys. Acta, Gen. Subj. 2006, 1760, 1248−1273. (25) Marmur, J. A Procedure for the Isolation of Deoxyribonucleic Acid from Microorganisms. J. Mol. Biol. 1961, 3, 208−211. (26) Allen, F. H. The Cambridge Structural Database: A Quarter of a Million Crystal Structures and Rising. Acta Crystallogr., Sect. B: Struct. Sci. 2002, 58, 380−388. (27) Grynpas, M.; Lindley, P. F. The Crystal and Molecular Structure of 1-Piperoylpiperidine. Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. 1975, 31, 2663−2667. (28) Bernstein, F. C.; Koetzle, T. F.; Williams, G. J. B.; Meyer, E. F., Jr.; Brice, M. D.; Rodgers, J. R.; Kennard, O.; Shimanouchi, T.; Tasumi, M. The Protein Data Bank: A Computer-based Archival File for Macromolecular Structures. J. Mol. Biol. 1977, 112, 535−542. (29) Coll, M.; Frederick, C. A.; Wang, A. H.; Rich, A. A Bifurcated Hydrogen-Bonded Conformation in the d(A.T) Base Pairs of the DNA Dodecamer d(CGCAAATTTGCG) and its Complex with Distamycin. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 8385−8389. (30) Coll, M.; Aymami, J.; Van der Marel, G. A.; Van Boom, J. H.; Rich, A.; Wang, A. H. Molecular Structure of the Netropsind(CGCGATATCGCG) Complex: DNA Conformation in an Alternating AT Segment. Biochemistry 1989, 28, 310−320. (31) Heinemann, U.; Alings, C.; Bansal, M. Double Helix Conformation, Groove Dimensions and Ligand Binding Potential of a G/C Stretch in B-DNA. EMBO J. 1992, 11, 1931−1939. (32) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E. UCSF Chimera-A Visualization System for Exploratory Research and Analysis. J. Comput. Chem. 2004, 25, 1605−1612. (33) Lang, P. T.; Moustakas, D.; Brozell, S.; Carrascal, N.; Mukherjee, S.; Pegg, S.; Raha, K.; Shivakumar, D.; Rizzo, R.; Case, D. A.; Shoichet, B.; Kuntz, I. D. DOCK, Version 6.2; University of California, San Francisco, USA, 2008. (34) Graves, A. P.; Shivakumar, D. M.; Boyce, S. E.; Jacobson, M. P.; Case, D. A.; Shoichet, B. K. Rescoring Docking Hit Lists for Model Cavity Sites: Predictions And Experimental Testing. J. Mol. Biol. 2008, 377, 914−934. (35) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; Roberts, B.; Hayik, S.; Roitberg, A.; Seabra, G.; Swails, J.; Goetz, A. W.; Kolossvai, I.; Wong, K. F.; Paesani, F.; Vanicek, J.; Wolf, R. M.; Liu, J.; Wu, X.; Brozell, S. R.; Steinbrecher, T.; Gohlke, H.; Cai, Q.; Ye, X.; Wang, J.; Hsieh, M. -J.; Cui, G.; Roe, D. R.; Mathews, D. H.; Seetin, M. G.; Salomon-Ferrer, R.; Sagui, C.; Babin, V.; Luchko, T.; Gusarov, S.; Kovalenko, A.; Kollman, P. A. AMBER 12; University of California: San Francisco, 2012. (36) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery, J. A., Jr.; Stratmann, R. E.; Burant, J. C.; Millam, J. M.; Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; K


Article

Journal of Chemical Information and Modeling Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian 98, Revision A.6; Gaussian, Inc.: Pittsburgh, PA, 1998. (37) Besler, B. H.; Merz, K. M., Jr.; Kollman, P. A. Atomic Charges Derived from Semiempirical Methods. J. Comput. Chem. 1990, 11, 431−439. (38) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. A WellBehaved Electrostatic Potential Based Method Using Charge Restraints For Determining Atom-Centered Charges: The RESP Model. J. Phys. Chem. 1993, 97, 10269−10280. (39) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. Development and Testing of a General AMBER Force Field. J. Comput. Chem. 2004, 25, 1157−1174. (40) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. A Second Generation Force Field for the Simulation of Proteins, Nucleic Acids, and Organic Molecules. J. Am. Chem. Soc. 1995, 117, 5179−5197. (41) Perez, A.; Marchan, I.; Svozil, D.; Sponer, J.; Cheatham, T. E., III; Laughton, C. A.; Orozco, M. Refinement of the AMBER Force Field for Nucleic Acids: Improving the Description of α/γ Conformers. Biophys. J. 2007, 92, 3817−3829. (42) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Fuctions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (43) Darden, T.; Perera, L.; Li, L.; Pedersen, L. New Tricks for Modelers from the Crystallography Toolkit: The Particle Mesh Ewald Algorithm and its Use in Nucleic Acid Simulations. Structure 1999, 7, R55−R60. (44) Darden, T.; York, D.; Pedersen, L. G. Particle mesh Ewald: An Nlog(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089−10092. (45) Feig, M.; Karanicolas, J.; Brooks, C. L., III MMTSB Tool Set; MMTSB NIH Research Resource, The Scripps Research Institute, 2001. (46) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual Molecular Dynamics. J. Mol. Graphics 1996, 14, 33−38. (47) Lu, X.-J.; Olson, W. K. 3DNA: A Software Package for the Analysis, Rebuilding and Visualization of Three-Dimensional Nucleic Acid Structures. Nucleic Acids Res. 2003, 31, 5108−5121. (48) Kollman, P. A.; Massova, I.; Reyes, C.; Kuhn, B.; Huo, S.; Chong, L.; Lee, M.; Lee, T.; Duan, Y.; Wang, W.; Donini, O.; Cieplak, P.; Srinivasan, J.; Case, D. A.; Cheatham, T. E., III Calculating Structures and Free Energies of Complex Molecules: Combining Molecular Mechanics and Continuum Models. Acc. Chem. Res. 2000, 33, 889−897. (49) Srinivasan, J.; Cheatham, T. E., III; Cieplak, P.; Kollman, P. A.; Case, D. A. Continuum Solvent Studies of the Stability of DNA, RNA, and Phosphoramidate-DNA Helices. J. Am. Chem. Soc. 1998, 120, 9401−9409. (50) Wang, J.; Morin, P.; Wang, W.; Kollman, P. A. Use of MMPBSA in Reproducing the Binding Free Energies to HIV-1 RT of TIBO Derivatives and Predicting the Binding Mode to HIV-1 RT of Efavirenz by Docking and MM-PBSA. J. Am. Chem. Soc. 2001, 123, 5221−5230. (51) Huo, S.; Wang, J.; Cieplak, P.; Kollman, P. A.; Kuntz, I. D. Molecular Dynamics and Free Energy Analyses of Cathepsin DInhibitor Interactions: Insight into Structure-Based Ligand Design. J. Med. Chem. 2002, 45, 1412−1419. (52) Luo, R.; David, L.; Gilson, M. K. Accelerated PoissonBoltzmann Calculations for Static and Dynamic Systems. J. Comput. Chem. 2002, 23, 1244−1253.

(53) Sitkoff, D.; Sharp, K. A.; Honig, B. Accurate Calculation of Hydration Free Energies Using Macroscopic Solvent Models. J. Phys. Chem. 1994, 98, 1978−1988. (54) Gholivand, M. B.; Kashanian, S.; Peyman, H.; Roshanfekr, H. DNA-Binding Study of Anthraquinone Derivatives Using Chemometrics Methods. Eur. J. Med. Chem. 2011, 46, 2630−2638. (55) Kelly, J. M.; Tossi, A. B.; McConnell, D. J.; OhUigin, C. A Study of the Interactions of some Polypyridylruthenium(II) Complexes with DNA Using Fluorescence Spectroscopy, Topoisomerisation and Thermal Denaturation. Nucleic Acids Res. 1985, 13, 6017−6034. (56) Tysoe, S. A.; Morgan, R. J.; Baker, A. D.; Strekas, T. C. Spectroscopic Investigation of Differential Binding Modes of.DELTA.and.LAMBDA.-Ru(bpy)2(ppz)2+ with Calf Thymus DNA. J. Phys. Chem. 1993, 97, 1707−1711. (57) Mati, S. S.; Roy, S. S.; Chall, S.; Bhattacharya, S.; Bhattacharya, S. C. Unveiling the Groove Binding Mechanism of a Biocompatible Naphthalimide-Based Organoselenocynate with Calf Thymus DNA: An “Ex Vivo” Fluorescence Imaging Application Appended by Biophysical Experiments and Molecular Docking Simulations. J. Phys. Chem. B 2013, 117, 14655−14665. (58) Stephanos, J. J. Drug-Protein Interactions: Two-Site Binding of Heterocyclic Ligands to a Monomeric Haemoglobin. J. Inorg. Biochem. 1996, 62, 155−169. (59) Zhong, W.; Wang, Y.; Yu, J.-S.; Liang, Y.; Ni, K.; Tu, S. The Interaction of Human Serum Albumin with a Novel Antidiabetic Agent SU-118. J. Pharm. Sci. 2004, 93, 1039−1046. (60) Sun, Y.; Ji, F.; Liu, R.; Lin, J.; Xu, Q.; Gao, C. Interaction Mechanism of 2-Aminobenzothiazole with Herring Sperm DNA. J. Lumin. 2012, 132, 507−512. (61) Nafisi, S.; Adelzadeh, M.; Norouzi, Z.; Sarbolouki, M. N. Curcumin Binding to DNA and RNA. DNA Cell Biol. 2009, 28, 201− 208. (62) Lakowicz, J. R.; Weber, G. Quenching of Fluorescence by Oxygen. Probe for Structural Fluctuations in Macromolecules. Biochemistry 1973, 12, 4161−4170. (63) Basu, A.; Kumar, G. S. Biophysical Studies on CurcuminDeoxyribonucleic Acid Interaction: Spectroscopic and Calorimetric Approach. Int. J. Biol. Macromol. 2013, 62, 257−264. (64) Haq, I. Thermodynamics of Drug−DNA Interactions. Arch. Biochem. Biophys. 2002, 403, 1−15. (65) Treesuwan, W.; Wittayanarakul, K.; Anthony, N. G.; Huchet, G.; Alniss, H.; Hannongbua, S.; Khalaf, A. I.; Suckling, C. J.; Parkinson, J. A.; Mackay, S. P. A Detailed Binding Free Energy Study of 2:1 Ligand-DNA Complex Formation by Experiment and Simulation. Phys. Chem. Chem. Phys. 2009, 11, 10682−10693. (66) Kumar, C. V.; Turner, R. S.; Asuncion, E. H. Groove Binding of a Styrylcyanine Dye to the DNA Double Helix: The Salt Effect. J. Photochem. Photobiol., A 1993, 74, 231−238. (67) Qiao, C.; Bi, S.; Sun, Y.; Song, D.; Zhang, H.; Zhou, W. Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe. Spectrochim. Acta, Part A 2008, 70, 136−143. (68) Bi, S.; Qiao, C.; Song, D.; Tian, Y.; Gao, D.; Sun, Y.; Zhang, H. Study of interactions of flavonoids with DNA using acridine orange as a fluorescence probe. Sens. Actuators, B 2006, 119, 199−208. (69) Mathew, K. V.; Unnikrishnan, N. V.; Sudarsanakumar, C. Molecular dynamics simulations and binding free energy analysis of DNA minor groove complexes of curcumin. J. Mol. Model. 2011, 17, 2805−2816. (70) Tabernero, L.; Verdaguer, N.; Coll, M.; Fita, I.; Van der Marel, G. A.; Van Boom, J. H.; Rich, A.; Aymami, J. Molecular Structure of the A-Tract DNA Dodecamer d(CGCAAATTTGCG) Complexed with the Minor Groove Binding Drug Netropsin. Biochemistry 1993, 32, 8403−8410. (71) Brown, D. G.; Sanderson, M. R.; Garman, E.; Neidle, S. Crystal structure of a berenil-d(CGCAAATTTGCG) complex: An example of drug-DNA recognition based on sequence dependent structural features. J. Mol. Biol. 1992, 226, 481−490. L


Article

Journal of Chemical Information and Modeling (72) Goodsell, D. S.; Ng, H. L.; Kopka, M. L.; Lown, J. W.; Dickerson, R. E. Structure of a dicationic monoimidazole lexitropsin bound to DNA. Biochemistry 1995, 34, 16654−16661. (73) Nguyen, B.; Neidle, S.; Wilson, W. D. A Role of Water Molecules in DNA−Ligand Minor Groove Recognition. Acc. Chem. Res. 2009, 42, 11−21. (74) Spackova, N.; Cheatham, T. E., III; Ryjacek, F.; Lankas, F.; Van Meervelt, L.; Hobza, P.; Sponer, J. Molecular Dynamics Simulations and Thermodynamics Analysis of DNA−Drug Complexes. Minor Groove Binding between 4′,6-Diamidino-2-phenylindole and DNA Duplexes in Solution. J. Am. Chem. Soc. 2003, 125, 1759−1769. (75) Varghese, M. K.; Thomas, R.; Unnikrishnan, N. V.; Sudarsanakumar, C. Molecular Dynamics Simulations of xDNA. Biopolymers 2009, 91, 351−360. (76) El-Barghouthi, M. I.; Jaime, C.; Akielah, R. E.; Al-Sakhen, N. A.; Masoud, N. A.; Issa, A. A.; Badwan, A. A.; Zughul, M. B. Free energy perturbation and MM/PBSA studies on inclusion complexes of some structurally related compounds with β-cyclodextrin. Supramol. Chem. 2009, 21, 603−610. (77) Xia, D.-H.; Ren, X.-D.; Jiao, L.; Li, H. Inclusion Modes of Berberine with β-Cyclodextrin in Aqueous Solution. Chem. Res. Chin. Univ. 2012, 28, 282−286.

M


Energetics, Thermodynamics, and Molecular Recognition of

Recommend Documents