Unveiling the Groove Binding Mechanism of a Biocompatible

Nov 8, 2013 - On account of having high antitumor activity toward various human and ... further modification of their structure according to pharamace...
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Unveiling the Groove Binding Mechanism of a Biocompatible Naphthalimide-Based Organoselenocyanate with Calf Thymus DNA: An “Ex Vivo” Fluorescence Imaging Application Appended by Biophysical Experiments and Molecular Docking Simulations Soumya Sundar Mati,† Somnath Singha Roy,‡ Sayantani Chall,† Sudin Bhattacharya,‡ and Subhash Chandra Bhattacharya*,† †

Department of Chemistry, Jadavpur University, Kolkata 700032, India Department of Cancer Chemoprevention, Chittaranjan National Cancer Institute, Kolkata 700026, India

‡

S Supporting Information *

ABSTRACT: The present study embodies a detailed investigation of the binding modes of a potential anticancer and neuroprotective fluorescent drug, 2-(5selenocyanato-pentyl)-6-chloro benzo[de]isoquinoline-1,3-dione (NPOS) with calf thymus DNA (ctDNA). Experimental results based on spectroscopy, isothermal calorimetry, electrochemistry aided with DNA-melting, and circular dichroism studies unambiguously established the formation of a groove binding network between the NPOS and ctDNA. Molecular docking analysis ascertained a hydrogen bonding mediated ‘A-T rich region of B-DNA’ as the preferential docking site for NPOS. The cellular uptake and binding of NPOS with DNA from “Ehrlich Ascites Carcinoma” cells confirmed its biocompatibility within tumor cells. Experimental and ex vivo cell imaging studies vividly signify the importance of NPOS as a potential prerequisite for its use in therapeutic purposes.

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INTRODUCTION Small molecules binding with DNA is of immense interest in the arena of medicinal or clinical chemistry.1,2 As a genetic instructor, DNA is involved in gene transcription,3 mutagenesis,4 gene expression,5,6 etc. and thus can be portrayed as one of the nature’s most elementary conduits for the development and functioning of living organisms.5 Therefore it has formed the nucleus of many-faceted research activities for years.5,6 Recognition and characterization of the interactions of small molecules with DNA is significantly fascinating as they give effective information for designing new and more efficient therapeutic agents in controlling gene expression.7,8 Okamoto et al.9 recently reported a fluorescence-controlled process for sensing nucleic acid using small molecule. It is evident from literature that diverse structural and electronic factors control the DNA binding affinity and sequence specificity of these small molecules.10,11 Thus, elucidation of the structure−function relationships and search for the key structural determinants mediating the mechanisms of action of drugs seem to be one of the most important problems in modern clinical research field. 12 In fact, selective, sensitive, and nondestructive physicochemical and particularly spectroscopic approaches facilitate determining the interaction of drugs with their target DNA and therefore, propose the ways for designing of new molecules with desired functions. In view of that urge for searching new and promising therapeutic agents, we herein introduced an anticancer drug 2© 2013 American Chemical Society

(5-selenocyanato-pentyl)-6-chloro benzo-[de]isoquinoline-1,3dione (NPOS) as a specific minor groove binder with DNA. Usually, drugs bind to DNA under the following three dominant modes of interactions: (1) intercalation, (2) groove, and (3) electrostatic. The groove binding involves docking of the thin ribbon-like molecules through hydrogen bonding or van der Waals interaction with the nucleic acid bases in the deep major groove or the shallow minor groove of the DNA helix.11,13 In this perspective, it is necessary to mention that the discovery of minor-groove recognition molecules such as distamycin and netropsin paved the way for considering the design and synthesis of DNA code-reading molecules that might ultimately target a unique sequence.14 Moreover, there are quite a few ligands, e.g., propamidine, berenil and furamidines which are known to bind selectively in the minor groove with specific base sequence of DNA thereby exerting their biological activity through the inhibition of DNAassociated enzymes such as DNA topoisomerases.15 Furthermore, there are certain DNA minor groove binding agents and a series of anticancer drugs which can proficiently block DNA helicase activity by binding to duplex DNA at specific base sequences.16 Received: September 10, 2013 Revised: November 6, 2013 Published: November 8, 2013 14655

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Ethidium bromide (EtBr) and ctDNA (molecular wt. 8.4 MDa) were procured from Fluka (Sigma) and used as received. Urea (CO(NH2)2, 99.5%), potassium iodide (KI, 99.8%), and N-[2hydroxyethyl] piperazine-N-[2-ethanesulphonic acid] (HEPES) buffer were obtained from SRL, India. All the experiments were performed using this buffer of 0.01 M (pH = 7). Spectroscopic grade 1,4-dioxane (Spectrochem, India) were used as received. Millipore water was used throughout the experiment. Stock solution of ctDNA was prepared by dissolving solid ctDNA in HEPES buffer (0.01 M) (pH = 7) and stored at 4 °C. The purity of ctDNA was verified by monitoring the ratio of absorbance at 260 nm to that at 280 nm, which was in the range 1.8−1.9. The concentration of ctDNA was determined spectrophotometrically using εDNA = 13600 M−1 cm−1 at 258 nm.13 The stock solution of compound NPOS (1.16 × 10−3 M) was prepared in a 1:1 dioxan−water solvent mixture.

On account of having high antitumor activity toward various human and murine cells, the research on functional 1,8naphthalimide derivatives as DNA targeting, anticancer and cellular imaging agents is a fast growing area and has resulted in several such derivatives entering into clinical trials.17 The rich photophysical properties of the naphthalimides (which are highly dependent on the nature and the substitution pattern of the aryl ring) make them prime candidates as probes as the changes in spectroscopic properties such as absorption, dichroism, and fluorescence can be used to monitor their binding to biomolecules. This also makes them useful species for monitoring their uptake and location within cells without the use of costaining. Depending on the substituents, naphthalimide derivatives have shown their selectivity toward A-T or G-C rich regions of DNA.18 Thus, such intriguing nature of naphthalimide derivatives highly stimulates the present work investigating the interaction of a antioxidant, anticancer and nontoxic naphthalimide derivative NPOS with ctDNA. In this report, we preferentially choose organoselenium compound of 1,8-naphthalimide since it is less toxic and could be used as enzyme inhibitors, neuroprotective, cancer chemo-preventive agents.19 As the synthesis and toxicity of this newly designed organoselenocyanate based derivative has already been reported in our previous work,19 hence exploring their biophysical properties and interaction with DNA is essentially important as the experimental outcome will further help to apply them toward clinical trial as a new generation drug molecule or assist any further modification of their structure according to pharamaceutical need. The information of NPOS-DNA interaction including tumor cell DNA sensitivity of the probe have been explained in this paper. Steady-state absorption, emission, circular dichroism along with time-resolved emission and rotational relaxation dynamics were employed to probe the strength, mode of binding, and dynamical aspects of the NPOSDNA interaction. Thermodynamic parameters and electrochemical measurements were also addressed for the binding affinity, binding stoichiometry, and electrical potential variation of DNA−NPOS interaction. Viscosity and helix melting study made obvious the progressive change in hydrodynamic and thermal stability of DNA bound probe. In addition, theoretical modeling was carried out to visualize the way and preferential docking position of NPOS in calf thymus DNA (ctDNA). Moreover ex vivo binding of DNA−NPOS was also investigated using DNA from tumor cells.

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METHODS Steady-State and Time-Resolved Spectral Measurements. UV−vis absorption spectra were obtained with a Shimadzu (model UV1700) spectrophotometer, and the absorbance versus temperature curves (melting profiles) of DNA and DNA−NPOS complex were measured on the same spectrophotometer well equipped with the peltier controlled accessory. The steady state fluorescence emission and excitation spectra were recorded on a Spex Fluorolog-2 spectrofluorimeter with an external slit width of 2.5 mm. All measurements were done repeatedly, and reproducible results were obtained. Time resolved fluorescence measurements were performed from time-resolved intensity decay by the method of time correlated single-photon counting using a picosecond diode laser at 370 nm (IBH Nanoled-03) as a light source with a TBX-04 detector (all IBH, UK). For time-resolved fluorescence anisotropy decay measurements, the parallel and perpendicular emission polarizations were controlled using polarizer. The data stored in a multichannel analyzer were routinely transferred to IBH DAS-6 decay analysis software. Viscometry. Viscosity measurements were made using an UBBELOHDE viscometer that was maintained at 25 ± 0.5 °C with a constant temperature bath. The volume of DNA solution was fixed at 8.0 mL, and flow time was measured with a digital stopwatch. To estimate the viscosity of the sample, the mean of three replicated measurements was taken. The values for relative specific viscosity (η/ηo)1/3, where ηo and η are the specific viscosity contributions of DNA in the absence and in the presence of NPOS, respectively, were plotted against r (r = [complex]/[DNA]). Electrochemical Studies. The electrochemical measurements were carried out with a BASi epsilon electrochemistry system. The electrochemical measurements were carried out with oxygen-free solutions made by purging with purified nitrogen. The reference electrode used was saturated Ag/AgCl/ KCl, which was isolated from the solution by salt bridge to prevent contamination through leakage from the electrode. The auxiliary and working electrodes were platinum foil and carbon paste electrode that were placed directly to the solution. The cyclic voltammetric (CV) measurement was carried out at 25 °C in buffer solution, and the concentration of the supporting electrolyte tetraethyl ammonium perchlorate (TEAP) was maintained at 0.1 M. All of the potentials reported in this study were referenced against the Ag/AgCl electrode. Pure ctDNA and blank solution are electrochemically inactive in the

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EXPERIMENTAL SECTION Materials. Naphthalimide derivative, (2-(5-selenocyanatopentyl)-6-chloro benzo-[de]isoquinoline-1,3-dione) (Structure 1) was synthesized using a described method elsewhere,19 and

it was purified by column chromatography. The compound was recrystallized using ethyl acetate-pet ether (1:1) before use. 14656

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chloride and 10 mM Trizma base, adjusted to pH 10.0. To it, 1% Triton X-100 was added just prior to use) and left overnight. On the next day, the slides were rinsed with phosphate buffer saline and stained with NPOS at a concentration of 10−3 M for 5 min. The slides were then washed and examined with a coverslip at 400× magnification under fluorescence microscope.

potential range of +0.2 to 0.0 V under our experimental conditions. Isothermal Titration Calorimetry. An OMEGA isothermal titration calorimeter (ITC) of Microcal, Northampton (U.S.A.) was used for thermometric measurements. During measurements, temperature was kept constant at 25 °C by circulating water from a Neslab RTE100 (U.S.A.) water bath maintained at 5 °C below the temperature of the calorimetric cell as per procedural requirement. The temperature in the calorimetric cells was accurate within ±0.01 °C. The heat released or absorbed at each step of dilution of DNA solution in buffer, and the enthalpy change per mole of injected probe was calculated with the help of the ITC software. The reproducibility was checked from repeated measurements. Circular Dichroism. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter using a rectangular quartz cuvette of path length 1 cm. The reported CD profiles are an average of four successive scans with 50 nm per minute scan time and an appropriately corrected baseline. The temperature was kept at 25 °C during the experiment. Molecular Docking Analysis. The crystal structures of ctDNA used for docking study were extracted from the structure having Protein Data Bank (PDB)20 identifier 3V9D (A-DNA) and 1BNA (B-DNA). Polar hydrogen atoms and Gasteiger charges were added to prepare the ctDNA molecule for docking analysis. Ligand docking was carried out applying the Lamarckian genetic algorithm (LGA) implemented in AutoDock 4.2.21 For docking of NPOS with ctDNA, the required file for the ligand (NPOS) was created through combined use of Gaussian 0322 program and AutoDock 4.2 software packages. The geometry of NPOS was first optimized at the Density Functional Theory23 using the B3LYP functional with the standard basis set, 6-31G(d,p), for all atoms using the Gaussian 03, and the resultant geometry was read in AutoDock 4.2 software in compatible file format. The grid size was set to 126, 126, and 126 along the x-, y-, and z-axis to recognize the binding site of NPOS in ctDNA, i.e., blind docking was performed. The lowest binding energy conformer was searched out of 20 different conformers for each docking simulation and the resultant one was used for further analysis. Other miscellaneous parameters were assigned to the default values obtained from the AutoDock program. The output from AutoDock was further analyzed with PyMOL software package.24 Cell Culture for Biodistribution of NPOS and Interaction of NPOS with DNA from Tumor Cells: Fluorescence Microscopy Study. Stock cells of Ehrlich Ascites Carcinoma (EAC) were cultured in RPMI-1640 and supplemented with 10% FBS, penicillin (100 IU mL−1) and streptomycin (100 μg mL−1) in a humidified atmosphere of 5% CO2 at 37 °C. At this condition the cells were seeded for 24 h in 6 well plates (2 × 105 cells in each well). The cell culture medium was then replaced with fresh medium containing the synthetic compound NPOS in a concentration of 10−3 M (1 molar stock solution of the probe was serially diluted with cell culture media to the required condition which consists of 0.1% DMSO) and incubated for 6 h at 37 °C in a 5% CO2/95% air atmosphere. Fluorescence images were taken at 400× magnifications under a fluorescence microscope (Model: Leica DM 4000B) with imaging system and an Ex/Em 350/ 422 nm filter. Primarily, EAC cells were layered over glass slide and immersed in a lysis solution (100 mM EDTA, 2.5 M sodium

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RESULT AND DISCUSSION Rationalization of Drug−DNA Binding: Steady-State and Time-Resolved Approach. Absorption and Emission. Quantitative evaluation of binding mode and the strength of the binding interaction between a small drug molecule (here NPOS) and DNA was investigated by employing steady-state absorption and fluorescence spectroscopy. The UV−vis absorption spectra of the probe were measured to assess the ground-state interaction of NPOS with nucleic acid. In aqueous buffer medium, NPOS shows a sharp lower-energy absorption band with a λabsmax at 350 nm. Addition of ctDNA to the buffer solution of NPOS causes appreciable decrease of absorbance without any shift of λabsmax (inset, Figure 1). This observation

Figure 1. Emission spectra of NPOS in presence of different concentrations of ctDNA. Curves (i)−(xii) correspond to 0.0, 0.025, 0.051, 0.101, 0.153, 0.204, 0.306, 0.408, 0.510, 0.663, 0.816, and 0.969 mM ctDNA, respectively. Inset: Absorption spectra of NPOS in the presence of different ctDNA concentrations of (i)−(xi). [NPOS] = 20 μM.

primarily indicated groove binding of the NPOS molecule with ctDNA, since it is a generally accepted fact that insignificant (or small) shift in absorption spectral behavior (i.e., λabsmax) is the most probable consequence of groove binding. By contrast, intercalation of small molecules into the DNA base stack is usually confirmed by the large shift of absorption maxima.4,13 The fluorescence spectrum of NPOS in aqueous solution shows a band at 422 nm upon excitation at 350 nm. Addition of ctDNA to the buffer solution of NPOS leads to the reduction of fluorescence intensity along with a slight blue shift of 10 nm (Figure 1). The observed decrease in fluorescence intensity points to the binding interaction between the probe and ctDNA. A quantitative rationalization of the drug−DNA binding strength is important in the evaluation of efficacy of the drug to function as a therapeutic agent.6 Therefore, to know the binding efficiency between NPOS-ctDNA in their respective ground and excited state, the observed absorbance and fluorescence intensities were quantified by plotting 1/ΔF as a 14657

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conformational stability of the biomacromolecules like DNA, proteins.6,11,27,28 With increasing concentration of the urea the variation of the relative fluorescence intensity (F/F0) of the DNA-bound NPOS is represented in Figure S1, which is reverse in pattern with respect to those observed during the fluorescence binding procedure. This inspection implies that urea-induced denaturation of DNA leads to considerable weakening of the binding and is able to release the fluorophore completely from the ctDNA environment. Fluorescence Quenching Study. The fluorescence quenching of NPOS in the ctDNA environment and in bulk aqueous buffer medium was studied using potassium iodide (KI) as a quencher. Quenching of the fluorescence of NPOS by KI in aqueous buffer solution and in the presence of 0.31 mM ctDNA was monitored using the Stern−Volmer equation (eq 2):29,30

function of ctDNA concentration using the Benesi−Hildebrand equation (eq 1)25 as follows to determine the binding constant: 1 1 1 1 = + K ΔFmax [L] ΔF ΔFmax (1) where ΔF = F0 − Fx and ΔFmax = F0 − F∞, and F0, Fx, and F∞ are the absorbance or fluorescence intensities of NPOS in the absence of ctDNA, at an intermediate ctDNA concentration, and at a concentration of complete interaction respectively; K is the binding constant, and [L] is the ctDNA concentration. The plot for elucidation of NPOS−ctDNA binding strength in relation to eq 1 shows (Figure 2a,b) the linearity with binding

F0 = 1 + KSV[Q ] (2) F where F0 is the fluorescence intensity without quencher, F is the quenched intensity of the fluorophore (NPOS), [Q] is the molar concentration of the quencher, and KSV is the Stern− Volmer quenching constant. Higher magnitude of KSV usually suggests more efficient quenching.6 Sahoo et al.13 in their earlier work also reported that in an aqueous solution, efficient iodide ion quenching (KSV values) was possibly due to groove binding. Compared with intercalative binding, groove binding exposes the bound molecules to the solvent surrounding the helix; as a result, the protection of the fluorophore from the ionic quencher in solution is expected to be in the order: intercalative binding > groove binding > electrostatic binding.31,32As observed earlier, the KSV values for intercalated probe into ctDNA account for the 6−8 times less compared to that in bulk water.6,32 In the present work, analysis of the quenching study (Figure 2C) shows appreciably high KSV values in an aqueous solution = 998 M−1 (±2.0%) and ctDNA = 657 M−1 (±2.5%) medium, which may be attributed to the groove binding of the probe to the DNA. Modulation of Dynamics and Rotational Relaxation Dynamics of NPOS upon Interaction with ctDNA: TimeResolved Fluorescence Decay and Time-Resolved Fluorescence Anisotropy Decay. Fluorescence lifetime measurement often serves as a sensitive indicator of the local environment of a fluorophore and is responsive toward excited state interactions.6 In an attempt to follow a generalized picture of the interaction, we have chosen to record the fluorescence

Figure 2. (a) Double reciprocal plot of 1/ΔFabs vs 1/[ctDNA] for the elucidation of the binding constant (K) between NPOS and ctDNA from absorption data. (b) Double reciprocal plot of 1/ΔFemis vs 1/ [ctDNA] for the elucidation of the binding constant (K) between NPOS and ctDNA from emission data. (c) Stern−Volmer plots for the fluorescence quenching of NPOS by KI in aqueous buffer, i.e., in the absence of ctDNA (pink) and in the presence of ctDNA environments (blue). [KI] × 105 = 1.6, 4.0, 6.4, 8.8, 13.6 M. [ctDNA] = 0.310 mM.

constant, K = 8.1 × 103 M−1 (±1.0%) (ground state) and K = 6.2 × 103 M−1 (±0.5%) (excited state). In comparison with literature reports for intercalative binding,26 this result indicates a much lower binding constant of the NPOS with DNA, and, hence, natural inquisition directs one to believe for a facile mode of groove binding for DNA-bound NPOS. The urea induced perturbation of the drug−DNA binding interaction was attempted as a complementary corridor to explore the binding phenomenon. Urea, being a well-known denaturant, can change the secondary and tertiary structure and

Figure 3. (a) Time resolved fluorescence decay of NPOS without and with ctDNA ([DNA] = 0.0, 3.7 × 10−5, 7.5 × 10−5, 15.1 × 10−5, 22.6 × 10−5, 30.1 × 10−5 M). (b) Temporal anisotropy decay curve of NPOS in the presence of ctDNA ([DNA] = 0.0, 7.5 × 10−5, 15.1 × 10−5, 22.6 × 10−5, 30.1 × 10−5 M). 14658

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than the fluorescence lifetime at the same concentration (Table 1), suggesting that the depolarization is essentially complete within the excited state lifetime of the probe in the specified environment. As emission lifetime is a time scale of the anisotropy decay,6,35 so in relation to the fluorescence lifetime of NPOS−ctDNA complex, the anisotropy of NPOS was also diminished with increasing ctDNA concentration. Sánchez et al.36 also reported a similar decrease of anisotropy value corresponding to the lifetime value. In either case, the observed time constants associated with the anisotropy decay of DNA− NPOS complex in the experimental DNA concentration range are in close agreement with the values reported for the minor groove binding in the DNA oligomer using other probes.37,38 Viscosity. Photophysical properties usually provide necessary but inadequate clues to support binding mode without hydrodynamic measurements (i.e., viscosity and sedimentation). A classical intercalation model demands lengthening of the DNA helix as base pairs are separated to accommodate the binding ligands, leading to increased DNA viscosity. By contrast, groove binding or electrostatic interaction typically causes less pronounced or only a minor change in the viscosity.39,40 In favor of this standpoint, the plot of relative specific viscosity (η/η0)1/3 versus the [complex]/[DNA] ratio (Figure 4a) for NPOS illustrates only a minor change in the viscosity in comparison with a classical DNA intercalator, ethidium bromide. Insignificant variation of relative specific viscosity of DNA in the presence of NPOS thus results in either electrostatic or groove binding mode of the probe. Cyclic Voltammetry. The application of electrochemical methods in the study of drug−DNA interactions endows with a useful complement to the previously used techniques. In the cyclic voltammetric (CV) study, the changes in the current and potential of NPOS without and with ctDNA are presented in Figure 4b. In the forward scan, a single anodic peak was observed which corresponds to the oxidation of NPOS (1.18 V), whereas in the reverse scan no cathodic peak was observed, indicating that the process is irreversible. From the figure it is observed that a regular increase in the anodic peak current with a shift of peak potential took place upon increasing the concentration of ctDNA. On addition of ctDNA, the peak potential of NPOS shifted from 1.18 V to 1.11 V. Sun et al.41 reported that the interaction of probe molecule with DNA resulted a significant increase of peak current, subsequently implying stronger interaction between DNA and probe.

decays of NPOS in the presence of varying concentrations of ctDNA. The fluorescence decay of NPOS in buffer solution was fitted as biexponential (Figure 3a) with time constants 0.92 and 5.85 ns. The data compiled in Table 1a reveal that with Table 1. (a) Time-Resolved Fluorescence Decay and (b) Time-Resolved Anisotropy Decay (r) Parameters of NPOS in Aqueous Buffer and DNA Environments (a) [DNA] (mM)

τ1 (ns)

τ2 (ns)

χ2

0.0 0.038 0.075 0.151 0.226 0.301 (b) [DNA] (mM)

0.92 0.91 0.90 0.88 0.87 0.86 τ1r (ns)

5.85 5.76 5.60 5.44 5.25 4.73 τ2r (ns)

1.14 1.02 1.22 1.15 0.99 1.01 χ2

0.0 0.075 0.151 0.226 0.301

0.90 0.96 0.99 1.00 1.01

6.00 4.22 4.16 4.14 3.75

1.12 1.09 1.21 1.11 1.25

increasing DNA concentration, the time-resolved fluorescence decay of the DNA bound probe decreases significantly to 0.86 and 4.73 ns. A lowering of lifetime of many fluorophores in macromolecule environment evinces the binding interaction between them.33,34 Consequently, here also the diminishing lifetimes of NPOS with ctDNA concentration substantiate the binding of NPOS−ctDNA. The time-dependent decay of fluorescence anisotropy is a sensitive indicator of the rotational motion or rotational relaxation of the fluorophore in an organized assembly.6 When a chromophore intercalates into the helix or in a DNA groove, its rotational motion should be restricted since it is rigidly held with a change of residence times on the time scale of the emission lifetime.35 NPOS exhibits biexponential anisotropy decay (with a reorientation time of 0.90 and 6.00 ns) in aqueous buffer, whereas in the presence of DNA, the decay changes drastically. The representative anisotropy decay profiles are given in Figure 3b, and the relevant rotational relaxation parameters are summarized in Table 1b. The rotational correlation time for NPOS in a particular DNA concentration is found to be less

Figure 4. (a) Effect of increasing amounts of EtBr and NPOS on the relative specific viscosity of ctDNA (150 μM). (b) Cyclic voltammograms of NPOS interaction with ctDNA. [ctDNA] = 0.0, 10.1 × 10−5, 25.5 × 10−5, 45.6 × 10−5 M. Scan rate: 300 mV S−1. Inset: Job plot for the complexation of NPOS with CT DNA. Change in fluorescence intensity of the free drug and the complex was monitored at the λmax of the complex. 14659

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Figure 5. Representative ITC profiles for the titration of (a) NPOS (2.5 mM) and (b) EtBr (2.5 mM) into a 0.25 mM solution of ctDNA at 20 °C. Each heat burst curve is the result of a 10 μL injection of NPOS into the DNA solution. The lower panels represent the corresponding normalized heat signals versus molar ratio. The data points (■) reflect the experimental injection heat, while the solid line represents the calculated fit of the data.

absorption or fluorescence intensity at λmax versus the mole fraction of NPOS exposed only a single binding mode both in ground and excited state. Mole fractions of NPOS at the intersection points are 0.55 and 0.57 (Figure S2 and Figure 4a inset), corresponding to stoichiometric ratios of 2:1 and 2:1 in the ground and excited states. Experimental observation from Job’s plot is in good agreement with the overall stochiometry determined employing ITC measurement. Effect of Ionic Strength. The change of ionic strength is an efficient method for distinguishing the electrostatic binding mode between small molecules and DNA.46 According to Figure S3, the fluorescence intensity of NPOS−ctDNA was scarcely amended after addition of NaH2PO4 (concentration range 0.00 to 0.06 M) into the system. If the interaction between NPOS and ctDNA is electrostatic, then Na+ partly neutralized the negative charges of the DNA phosphate backbone. Consequently, NaH2PO4 would certainly weaken the quenching effect of nucleic acids to the NPOS fluorescence and the fluorescence of NPOS-nucleic acid system must be enhanced along with the increase of the NaH2PO4 concentration.47 Here the experimental sequence rules out the electrostatic binding and sorted the NPOS−ctDNA interaction to groove binding. Circular Dichroism Study. The conformational changes of DNA associated with the small molecule binding can be studied efficiently by CD spectroscopy during the drug−DNA interactions. The CD spectra of the ctDNA duplex displayed a canonical B-form conformation characterized by a positive band at 273−280 nm due to base stacking and a negative band at 245 nm due to right handed helicity.10,48 To ascertain the NPOS-induced changes in the DNA conformation, the intrinsic CD profile of ctDNA in the far-UV (200−350 nm) wavelength region was recorded in the presence of varying [NPOS] (Figure 6). CD spectra show insignificant change in the case of minor groove binding and electrostatic binding, whereas the intercalative binding affects both the positive and negative bands.11,48 As illustrated in Figure 6, it reveals that both bands of the CD spectrum of ctDNA are significantly unchanged with increasing concentration of NPOS, indicating that the binding of the probe with ctDNA does not disturb the stacking of the bases. This observation optimistically rules out intercalation of

Furthermore, according to Devi et al.,42 with increasing concentration of ctDNA, the anodic peak potentials of the compound shifted toward lower values, indicating the nonintercalative binding nature of the complex with ctDNA. Isothermal Titration Calorimetry. To gain information from the thermodynamics of the DNA binding process, the interaction of NPOS with ctDNA was investigated using ITC. Moreover, it can provide noteworthy insight into the energetics and thermodynamic characterization of the binding interaction between the molecules and DNA.43,44 Figure 5 illustrates the heats of reaction plotted against the mole ratio of the probe to ctDNA after the baseline correction. The binding between NPOS−ctDNA was portrayed by exothermic reaction. The binding constant and thermodynamic parameters of NPOS−ctDNA complex estimated by fitting the integrated heats, are Kb = 12.7 × 103 M−1 (±2.5%), ΔH = −2.1 × 101 kcal mol−1 (±2%), and n = 0.43. ‘n’ is the site size of NPOS binding, which is the reciprocal of the overall stoichiometry (i.e., 2) of NPOS binding with ctDNA. However, these values are quite different in comparison to the wellknown intercalator EtBr−ctDNA complex; Kb = 2.52 × 105 M−1 (±2%), ΔH= −1.1 × 101 kcal mol−1 (±2%). Again, the favorable free energy changes (ΔG = −5.61 kcal mol−1) calculated from NPOS−ctDNA binding constant along with large negative enthalpy changes make plain that binding of ctDNA with the NPOS molecules is enthalpy driven. Though the structural differences are reflected in the variation of thermodynamic parameters45 but the change in binding constant, enthalpy, and free energy between NPOS−ctDNA and EtBr−ctDNA can be conjectured as two different modes of binding with ctDNA. Earlier, in the work of Tjahjono et al.,45 they distinguished the meso-tetrakis derivatives as groove and/ or intercalative binder with ctDNA depending on the binding constant values and thermodynamic parameters. In the present work, therefore, the thermodynamic outcomes from calorimetry experiments convey that the binding of NPOS with ctDNA is a favorable process with negative enthalpy to the groove of ctDNA. Binding Stoichiometry (Job Plot). Continuous variation analysis procedure (Job plot) was employed using absorbance and fluorescence intensity to establish the binding stoichiometry of NPOS with the DNA.14 The plot of difference in 14660

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increase in Tm of ctDNA. Inappreciable change in Tm for ctDNA conformity supports the groove binding between NPOS and ctDNA. Molecular Docking Analysis. To understand the efficiency of a biologically active drug molecule to function as a therapeutic agent, the knowledge of its binding location in the DNA environment is very essential and significant. A docking experiment was performed to obtain an insight of selected binding site along with the preferred orientation of the ligand inside the DNA groove.40,52 Moreover, the docking study further corroborated the existence of interactions between the NPOS and DNA. The NPOS−DNA complex could fit well into the minor groove of the DNA with a binding site of three base pairs, preferentially involving the G-C residues (A-DNA) (Figure 8a) and A-T residues (B-DNA, which is the normal form of ctDNA) as revealed by the docked structure (Figure 8b). Figure 8b,c indicates that the mode of action of NPOS holding three base pairs is like A-T, A-T, G-C (B-DNA) and A-T, G-C, G-C (A-DNA) to the outer surface, i.e., the groove of the DNA biomolecule. Thus, the A-T regions seem to facilitate a better fit of the NPOS molecule into the minor groove of B-DNA, whereas for A-DNA it appears to G-C regions probably as a result of van der Waals’ interactions with the functional groups defining the groove. In addition, there are hydrogen bonding interactions of 37-O and 38-O of NPOS with H atoms of the corresponding base pairs. The lengths of hydrogen bond with guanine and cytosine bases (Figure 8c) are 1.83 and 2.66 Å, respectively, for A-DNA. For B-DNA, the lengths of hydrogen bond with adenine bases are 2.22 and 3.03 Å. The energy of the minimized docked structure is −4.85 kcal M−1 for A-DNA and −5.41 kcal M−1 for B-DNA, which is the best possible geometry of the NPOS inside the corresponding DNA minor groove. Moreover, substituted naphthalimides have shown their selectivity toward both A-T-rich or G-C-rich regions of DNA.18 Drug molecules preferably binds to the minor groove of the A-T-rich region of B-DNA (ctDNA).52 Thus the energy and binding constant value (Kb = 4.04 × 103 for A-DNA and 9.07 × 103 for B-DNA) also confirms the binding between these NPOS and B-DNA in preference to the A-T-rich region. The aromatic ring structure of NPOS having a long chain single bond allows for torsional rotation in order to fit into the narrower helical curvature of the minor groove. In the final docked conformation, the NPOS molecule exists in a crescent shape, which harmonizes with the natural curvature of the minor groove of DNA. The radius of the curvatures was calculated to be 6.60 Å and 6.49 Å for A-DNA and B-DNA, respectively. Cellular Uptake of NPOS and Its Interaction with Cellular DNA from Tumor Cells: An Ex Vivo Approach. Fluorescence property of a compound allows us to examine the cellular uptake and intracellular distribution of that compound by fluorescence microscopy. Here tumor cells were incubated with NPOS or ethidium bromide (used as a standard compound) at a concentration of 10−3 M for 6 h. Ethidium bromide was used since it is widely accepted for its fluorescence and DNA intercalating property. We observed bright fluorescence intensity spread all over the cells incubated with NPOS (Figure 9b) and ethidium bromide (Figure 9c). Most importantly, the deep spots around the nuclei were also observed (indicated by arrow). By contrast, no fluorescence intensity was visualized in the control cells (incubated without the compounds) (Figure 9a). As shown in Figure 9b, the fluorescence intensity appeared

Figure 6. Representative CD spectrum of ctDNA resulting from the interaction of 0.0, 1.6, 2.4, 4.0, 5.6, and 7.2 μM of NPOS.

NPOS in the DNA helix and thereby implies that the fluorophore binds to the host DNA through groove binding. Helix Melting Study. DNA melting is the process of separating the double helical DNA structure into two single strands from stable hydrogen bonding and base stacking interaction.49 With increasing temperature, the double helix structure of DNA is separated to a single strand as heat spoils the forces. The melting temperature (Tm) of DNA is defined as the temperature at which half of the DNA strands are in the double-helical state and half in the random-coil state.50 Helix melting of DNA is performed by measuring the absorbance at 260 nm as a function of temperature. The extinction coefficient of DNA bases at 260 nm in the double-helical form is much less than in the single stranded form. Hence, the absorbance increases piercingly as the helix melts and the DNA strands separate.51 Large increase in melting temperature (3−8 °C) is observed only for the strong intercalation type of interaction. On the contrary, groove-binding interaction of small molecules with DNA leads to insignificant amendment in Tm.6,39 In this case, experiment was carried out to monitor the change in Tm for ctDNA in the absence and presence of NPOS to follow up the specific groove binding between them (Figure 7). From the DNA melting profile, the estimated melting temperatures appear to be 66.2 (±0.3)°C, and 68.1 ((±0.3)°C for the unbound ctDNA and NPOS bound DNA, respectively. The melting curve was followed the reversibility in nature with very slow cooling. Binding of NPOS does not lead to a significant

Figure 7. Thermal melting profiles (absorbance change at 260 nm versus temperature) of ctDNA (50 μM) (black) and its complex with NPOS (25 μM) (red). 14661

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Figure 8. Docked pose of NPOS bound to the minor groove of DNA: (a) A-DNA; (b) B-DNA. (c) Close-up view of NPOS−A-DNA binding with two intermolecular H-bonds shown in yellow.

DNA conferred blue fluorescence image due to the DNA interaction (Figure 9e).

to be spread all over the cells, but more intensive spots indicated an accumulation of the target compounds in cell compartments such as the nucleus; a similar effect was also reported by Ott et al.53 Thus, by inspecting Figure 9b it can be concluded that the compound was taken up into the cells, and it trafficked mostly to compartments located around the nuclei (shown by the arrows). This was even confirmed by the fluorescence image of the cellular DNA incubated with NPOS (Figure 9e) and ethidium bromide (Figure 9f). Again owing to the interaction of DNA with NPOS and ethidium brimode, both became bound with the cellular DNA and this was proved by the appearance of fluorescence images of the DNA after staining with NPOS and ethidium bromide (Figure 9e,f). By contrast, the cellular DNA without the compound had no fluorescence intensity (Figure 9d). Actually, the lysis solution consisting of high salts and detergents disrupt the all cellular compartments and liberate the DNA. Here, the interaction of the NPOS with the liberated

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CONCLUSION Summing up, this work unveils the hitherto unknown interaction of bioactive selenocyanide 1,8-naphthalimide molecule with calf thymus DNA. The string of experimental and theoretical results basically indicates a predominantly grooved type binding between NPOS and ctDNA. Furthermore, it was observed that NPOS exhibits considerable ability to bind with the A-T-rich sequence of DNA, preferably in the minor groove. Finally, in view of the vast area of biological research, this report has successfully widened the scope for using the drug NPOS as a promising DNA binder with further scope for development. The results, reported herein, undoubtedly enhance the prospective probability of using NPOS as an efficient DNA binder and also encourage further development of NPOS analogous compounds in future for relevant pharmacological applications. 14662

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Figure 9. Fluorescence image of Ehrlich Ascites Carcinoma cells. (a) Control cells incubated without compounds. (9b) Cells exposed to 10−3 M of NPOS for 6 h. (9c) Cells exposed to 10−3 M of ethidium bromide for 6 h (arrow indicates dark spots). (d) DNA of cells without the compounds. (e) Cellular DNA interacted with NPOS for 5 min. (f) Cellular DNA interacted with ethidium bromide for 5 min.

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Cytotoxicity of Certain Mixed-ligandruthenium(II) Complexes of 2,2′dipyridylamine and Diimines. Dalton Trans. 2008, 16, 2157−2170. (3) Ma, Y.; Zhang, G.; Pan, J. Spectroscopic Studies of DNA Interactions with Food Colorant Indigo Carmine with the Use of Ethidium Bromide as a Fluorescence Prob. J. Agric. Food Chem. 2012, 60, 10867−10875. (4) Fei, Y.; Lu, G.; Fan, G. Spectroscopic Studies on the Binding of a New Quinolone Antibacterial Agent: Sinafloxacin to DNA. Anal. Sci. 2009, 25, 1333−1338. (5) Li, X. L.; Hu, Y. J.; Wang, H.; Yu, B. Q.; Yue, H. L. Molecular Spectroscopy Evidence of Berberine Binding to DNA: Comparative Binding and Thermodynamic Profile of Intercalation. Biomacromolecules 2012, 13, 873−880. (6) Paul, B. K.; Guchhait, N. Exploring the Strength, Mode, Dynamics, and Kinetics of Binding Interaction of a Cationic Biological Photosensitizer with DNA: Implication on Dissociation of the Drug− DNA Complex via Detergent Sequestration. J. Phys. Chem. B 2011, 115, 11938−11949. (7) Alniss, H. Y.; Anthony, N. G.; Khalaf, A. I.; Mackay, S. P.; Suckling, C. J.; Waigh, R. D.; Wheate, N. J.; Parkinson, J. A. Rationalising Sequence Selection by Ligand Assemblies in the DNA Minor Groove: The Case for Thiazotropsin A. Chem. Sci. 2012, 3, 711−722. (8) Ketron, A. C.; Denny, W. A.; Graves, D. E.; Osheroff, N. Amsacrine as a Topoisomerase II Poison: Importance of Drug−DNA Interactions. Biochemistry 2012, 51, 1730−1739. (9) Okamoto, A. Excitonic Interaction: Another Photophysical Process for Fluorescence-Controlled Nucleic Acid Sensing. Chem. Rec. 2010, 10, 188−196. (10) Bhowmik, D.; Hossain, M.; Buzzetti, F.; D’Auria, R.; Lombardi, P.; Kumar, G. S. Biophysical Studies on the Effect of the 13 Position Substitution of the Anticancer Alkaloid Berberine on Its DNA Binding. J. Phys. Chem. B 2012, 116, 2314−2324. (11) Jana, B.; Senapati, S.; Ghosh, D.; Bose, D.; Chattopadhyay, N. Spectroscopic Exploration of Mode of Binding of ctDNA with 3-

ASSOCIATED CONTENT

S Supporting Information *

Figure S1: Plot of the variation of the relative fluorescence intensity (F/F0) of the NPOS bound ctDNA as a function of increasing concentration of urea. Figure S2: Job plot for the binding of NPOS with CT DNA. Change in absorption of the free drug and the complex was monitored at the λmax of the complex. Figure S3: The effect of NaH 2 PO 4 on the fluorescence of NP-ctDNA; [NP] = 20 μM; [ctDNA] = 0.510 mM; [NaH2PO4]: 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 mM. This information is available free of charge via the Internet at http://pubs.acs.org

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; scbhattacharyya@chemistry. jdvu.ac.in. Phone No: 033 2414 6223/Fax: 91(033) 24146584. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Author S.S.M. thanks UGC for providing SRF. One of the authors, S.C. acknowledges CSIR for providing SRF. Author S.B. gratefully acknowledges CSIR for the financial assistance. S.S.R. thanks CSIR for the Fellowship.

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