Spectroscopic and Thermodynamic Insights into the Interaction

Article pubs.acs.org/JPCB

Spectroscopic and Thermodynamic Insights into the Interaction between Proflavine and Human Telomeric G‑Quadruplex DNA Vivek Kumar,† Abhigyan Sengupta,† Krishna Gavvala, Raj Kumar Koninti, and Partha Hazra* Department of Chemistry, Indian Institute of Science Education and Research (IISER)-Pune, Pune 411008, Maharashtra India ABSTRACT: The G-quadruplex (GQ-DNA), an alternative structure motif of DNA, has emerged as a novel and exciting target for anticancer drug discovery. GQ-DNA formed in the presence of monovalent cations (Na+/K+) by human telomeric DNA is a point of interest due to their direct relevance for cellular aging and abnormal cell growths. Small molecules that selectively target and stabilize G-quadruplex structures are considered to be potential therapeutic anticancer agents. Herein, we probe G-quadruplex and proflavine (a well-known DNA intercalator, hence acting as an anticarcinogen) association through steady state and time-resolved fluorescence spectroscopy to explore the effect of stabilization of GQ-DNA by this well-known DNA intercalator. The structural modifications of G-quadruplex upon binding are highlighted through circular dichroism (CD) spectra. Moreover, a detailed insight into the thermodynamics of this interaction has been provided though isothermal titration calorimetry (ITC) studies. The thermodynamic parameters obtained from ITC help to gain knowledge about the nature as well as the driving forces of binding. This present study shows that proflavine (PF) can act as a stabilizer of telomeric GQ-DNA through an entropically as well as enthalpically feasible process with high binding affinity and thereby can be considered as a potential telomerase inhibitor.

1. INTRODUCTION Living in an era of increasing cancer threats, one of the major focuses of modern science is anticancer drug development and its application toward a hazard free mankind. Numerous drugs are known either to intercalate or bind to groove which results in inhibition for DNA replication and hence cell division. Not only the canonical duplex DNA but also several noncanonical DNA structures are treated as potential targets for anticancer drugs.1−4 Among various noncanonical DNA motifs, a special kind motif known as G-quadruplex (GQ-DNA) has demanded burgeoning attention since the discovery of the tetramer guanosine residue by Gellert et al. in 1962.5 G-quadruplex is a four-stranded DNA structure, comprising stacked G-quartets held together by eight Hoogsteen hydrogen bonds.1−6 The formation of GQ-DNA is observed in G-rich DNA sequences at the human telomeric end and in the promoter region of some proto-oncogens.7−9 At the ends of the chromosomes, i.e., the telomeres, DNA is devoid of a complex protein-coding sequence. Human telomeres are usually formed by 5′TTAGGG-3′ sequence repeats.2 The bulk of telomeric DNA adopts a double-helical conformation by pairing a G-rich sequence with a C-rich complementary sequence. However, at the 3′ end of such telomeric DNA resides overhang, as single stranded G-rich DNA of approximately 100−200 bases in length.10 This single stranded DNA has a very high affinity to fold into a G-quadruplex structure in the presence of small cations such as Na+, K+, etc.11,12 Typically, Na+ induces an antiparallel structure, while K+ induces a mixed population of both parallel and antiparallel G-quadruplex structures of human telomeric DNA6,12 which can inhibit the replication of the quadrplex sequence.11,12 It is believed that telomere plays an important role in cellular aging and cancer,13 and therefore, © 2014 American Chemical Society

telomeric G-quadruplex structures have received widespread attention. Keeping focus over the inhibition of telomerase activity by GQ-DNA, recent research explored several small molecules that can interact with telomeric DNA and effectively inhibit telomerase activity.14−16 These small molecules and/or ligands that recognize and bind to GQ-DNA in telomeric DNA are celebrated as potential anticancer drugs.6,14−20 In general, three important structural features are required for a molecule to be considered as a GQ-DNA binder. The molecule should contain (a) a heteroaromatic ring capable of π-staking interaction with nucleobases, (b) a planner structural part, which can fit over Gquartet plane, and (c) a cationic center, which can interact with phosphate backbone of DNA through electrostatic interaction.17,21Among various such kinds of molecules, proflavine is known to have a mutagenic effect through intercalation between nucleic acid base pairs in double stranded DNA. Proflavine (acridine-3,6-diamine), an acridine derivative, belongs to the class of polynuclear N-heteroaromatic family and has a number of significant pharmaceutical uses.22,23 The intercalation of acridine and its derivatives prevents DNA replication in rapidly growing cancer cells.24−27 Therefore, many of the acridine derivatives are well used in chemotherapeutic agents.28 There are some reports regarding acridine derivatives interacting with G-quadruplex DNA.29−32 Interestingly, studies related to interaction of proflavine (PF), believed to be a precursor of all reported acridine derivatives, with GQDNA is not available. Looking at the structure of PF (Scheme 1), there is a high possibility that PF can interact and provide Received: June 24, 2014 Revised: September 1, 2014 Published: September 2, 2014 11090

dx.doi.org/10.1021/jp506267b | J. Phys. Chem. B 2014, 118, 11090−11099

The Journal of Physical Chemistry B

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JobinYvon IBH, U.S.A.). For Job’s plot, concentrations of PF and GQ-DNA were fixed at 4.5 μM and the fluorescence was measured at respective emission maximum with varying mole fraction of PF. Then, the change in intensity was plotted against mole fraction of PF and from the break point of the plot the complexation ratio was determined. In TCSPC, PF molecules were excited using 440 nm diode laser (nano-LED 440, fwhm ∼140 ps) and the emission photons at respective emission maximums were collected at magic angle using a MCP-PMT (Hamamatsu, Japan) detector. For time-resolved anisotropy study, we have used a motorized polarizer in the emission side. The emission intensities at perpendicular (IVH(t)) and parallel polarization (IVV(t)) were collected alternatively for 60 s. For typical anisotropy decay, the difference between the peak counts at parallel and perpendicular polarization was kept to 3000. The G factor for the instrument was measured independently by using a horizontally polarized excitation beam and measuring the two perpendicularly polarized fluorescence decays. The analysis of lifetime and anisotropy was done by IBH DAS6 software. We fitted both lifetime as well as anisotropy data with minimum number of exponential. Quality of each fitting was judged by χ2 values and the visual inspection of the residuals. The value of χ2 ≈ 1 was considered as best fit for the plots. Circular dichroism (CD) spectra were recorded on a J-815 CD (JASCO, U.S.A.) instrument at 25 °C. The data were collected at 1 nm intervals with 1 nm bandwidth. All measurements were taken in 0.2 cm path length cuvette with 400 μL sample volume. Each CD profile is an average of 3 scans of the same sample collected at a scan speed 100 nm/min, with a proper baseline correction from the blank buffer. During CD measurement, GQ-DNA concentration was kept fixed and the concentrations of PF was increased steadily. However, for induced CD measurement PF concentration was kept fixed and the concentration of H24-DNA (single strand) was increased steadily. To ensure equilibration, each spectrum was recorded after 10 min of the addition of H24-DNA. Isothermal titration calorimetry (ITC) measurements were performed in iTC200 microcalorimeter (Microcal-200) at 25 °C. All samples and the buffer were degassed prior to use. The titration of G-quadruplex against PF was performed in 20 injections (2 μL of each) into a solution with fixed GQ (200 μL) concentration in the cell with 200 s resting time between two consecutive injections. A blank experiment was also performed by injecting the same concentration of PF into PBS buffer under identical experimental condition and was used to correct dilution effect. The isotherm was analyzed using single-site binding model and nonlinear least-squares fitting algorithm built-in Microcal LLC ITC software to yield the relevant thermodynamic parameter. Melting study was performed using Varian Cary 300 Bio UV−vis Spectrophotometer (Thermo Fisher Scientific, U.S.A.). Data were analyzed by using Origin-Pro 8 software. Thermal melting was monitored at 290 nm with a heating rate of 1 °C/ min. Here we have provided melting temperatures (Tm) from the best sigmoidal curve fit of the melting profile.

Scheme 1. Different Prototropic Forms of Proflavine (PF)

additional stability to GQ-DNA, as it has (i) a heteroaromatic ring capable of π-staking interaction with nucleobases, (ii) a planner structure, which can fit over G-quartet plane, and (iii) a cationic center which is necessary to involve electrostatic interaction with phosphate backbone of DNA.17 Herein, we have probed GQ-DNA and PF interaction through steady state and time-resolved fluorescence spectroscopy to explore the interaction scenario and hence the extent of stabilization of GQ-DNA by an extrinsic noncovalent fluorescent marker, PF. In this study, 24-mer human telomeric (H24) sequence is used for the formation GQ-DNA. The structural modifications of GQ-DNA upon binding with PF are highlighted through circular dichroism (CD) studies. A very detailed insight into the thermodynamics of interaction has been provided through isothermal titration calorimetry (ITC) studies. The thermodynamic parameters help to gain knowledge about the nature as well as driving forces of the binding. The present work shows that PF can act as a GQ-DNA stabilizer through entropically as well as enthalpically feasible processes with high binding ability, and therefore, it can be considered as a potential telomerase inhibitor.

2. MATERIALS AND METHODS The 24-mer human telomeric (H24) DNA (5′-TTAGGGTTAGGGTTAGGGTTAGGG-3′) was purchased from Integrated DNA Technologies (IDT) and used as received. Stock DNA solution was prepared by dissolving it in 5 mM KH2PO4 and 5 mM K2HPO4 (pH 6.8) containing 150 mM KCl (PBS). All experiments and sample preparations were carried out in autoclave Millipore water. Before experiments H24-DNA in PBS was annealed at 90 °C for 10 min and stored at 4 °C for 48 h. For induced CD experiment, H24-DNA in Millipore water was annealed at 90 °C for 10 min and subsequent cooled at 0 °C. The concentration of GQ-DNA formed from H24-DNA was determined using the molar extinction coefficient of ∼244 600 M−1 cm−1 at 260 nm provided by IDT, USA. Here it is necessary to mention that IDT has determined the molar extinction coefficient using nearest neighbor approximation model. Before doing any experiment, the formation of (3 + 1) hybrid G-Quadruplex (GQ-DNA) structure was confirmed through CD. PF was purchased from Sigma-Aldrich and used without further purification. The PF was dissolved in the same buffer and concentration was determined by the UV−visible spectrophotometer (Shimadzu UV-2600) using the molar extinction coefficient33 41 000 M−1 cm−1. Absorbance measurements were performed in Shimadzu UV2600 UV−vis. Spectrophotometer, and steady-state fluorescence spectra were recorded in FluoroMax-4 spectrofluorimeter (Horiba Scientific, U.S.A.). Steady state anisotropy (r) is calculated using the following equation: I − I⊥ r= I + 2I⊥ (1)

3. RESULTS AND DISCUSSION 3.1. Steady State Results. PF in buffer (PBS, pH 6.8) shows a single unstructured absorption band at ∼444 nm (Figure 1a), which is believed to be originated from the π−π* transition of PF in aqueous medium. This observed spectrum is in good agreement with the previous reports.34 Increasing

where I∥ and I⊥ are the intensities measured at parallel and perpendicular to the electric vector of polarized incident light. Time-resolved fluorescence decays were collected using time correlated single photon counting (TCSPC) setup (Horiba 11091



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one of the major reason for the observed quenching. In this ET process, nucleobases (adenosine (Eox = 1.42 eV),35,36 cytidine (Eox = 1.6 eV),35 guanosine (Eox = 1.29 eV),35 and thymidine (Eox = 1.7 eV)36) act as potential electron donor and photoexcited PF (Eox = −0.78 V)37 acts as an effective electron acceptor. Ground state complex formation or static quenching may also contribute toward this quenching, as the absorption spectra indicate a strong ground state interaction between PF and GQ-DNA (Figure 1a). Here, the possibility of resonance energy transfer for possible reason for fluorescence quenching can be easily ruled out, since there is no overlap between the emission spectra of PF (donor) and the absorption spectrum of any of the nucleobases (acceptor). We believe that the interaction between protonated nitrogen of PF and negative phosphate groups of GQ-DNA is an important step toward the association process between GQ-DNA and PF. A very similar view of PF intercalation in wild DNA is recently reported by Wilbee et al.38 Notably, electron transfer from nucleobases to PF leading to 80% quenching needs a very strong stacking interaction between molecules, which is possible either by intercalation in between quartet or by stacking interaction at the top or bottom of the GQ-DNA, because simple groove binding cannot promote this kind of severe quenching. Moreover, it is verified that the energetic for opening up an intercalation site which is embedded within a stack of Gquartets is unfavorable.39 Therefore, we believe that PF does not intercalate but involves π-type stacking interaction with Gquartets at the two ends of GQ DNA. This concept is also supported by earlier reports with a range of ligands and quadruplexes.1,3,4,40,41 Although steady-state studies offer a notion about the interaction between PF and GQ-DNA, it cannot provide explicit picture of the interaction. Thus, we have used other techniques, like, time-resolved fluorescence, CD measurements, ITC, and Job’s plot (discussed later in this manuscript) to provide the molecular picture of interaction between PF and GQ-DNA. The association or binding constant is calculated by plotting the fluorescence changes (Figure 2a) with concentration of GQ-DNA using the following equation:42

Figure 1. Absorption (a) and fluorescence (b) spectra of PF (4.5 μM) in PBS (pH 6.8) with increasing GQ-DNA concentrations (in μM); where, i → vii stands for 0, 0.25, 0.5, 3.5, 5, 10, and 13.5. Inset in (a) shows shift with increasing concentration of GQ-DNA (μM); (i) 0, (ii) 2.5, (iii) 13.5, and inset in (b) shows intensity vs concentration of GQ-DNA plot.

⎛ ΔF ⎞2 ⎛ ΔF ⎞ C 0⎜ ⎟ − (C0 + Cp + Kd)⎜ ⎟ + Cp = 0 ⎝ ΔFmax ⎠ ⎝ ΔFmax ⎠

concentration of GQ-DNA results in significant decrease in absorption (almost 53%) along with a bathochromic shift from 444 to 457 nm (up to maximum addition of 13.5 μM GQDNA). The reduced absorption along with ∼13 nm red shift infers a possible switch of polarity around PF molecules due to the binding interaction with GQ-DNA. The red shift suggests that PF senses a less polar environment, as it is well established that higher polarity around the PF vicinity leads to a strong blue shift in absorption spectrum.34 The significant alteration of absorption spectra may be the first indication of the interaction between PF and GQ-DNA. The room-temperature emission spectra of PF in pH 6.8 show a single unstructured band with the emission maximum around 512 nm (Figure 1b), which is believed to be originated from the π−π* singlet state.34 With the gradual addition of GQ-DNA, emission spectra exhibit blue shift along with severe quenching effect (∼80% at 13.5 μM of GQ-DNA), implying a strong interaction between PF and GQ-DNA. The blue shift is attributed to the reduced polarity experienced by PF molecules during interaction with GQ-DNA, as it is reported that reduced polarity of the solvents leads to blue shift in emission spectra of PF.34 Electron transfer (ET) from nucleobases to PF, which promotes nonradiative decay pathways, may be attributed to

(2)

where C0 is the initial concentration of PF, ΔF is change in fluorescence intensity at 510 nm after addition of each aliquot of GQ-DNA, ΔFmax is change in fluorescence intensity when PF is totally bound to GQ-DNA, and Cp is the concentration of GQ-DNA added. The binding constant (Ka = 1/Kd) is determined to be 2.4(±0.4) × 106 M−1 from the nonlinear least-square analysis (using IGOR Pro software) of eq 2. The estimated binding constant is in good agreement with the observed binding affinity of alkaloids and other drugs to GQDNA.17,43,44 Anisotropy measurements can provide insight about an association or binding phenomenon,45 and we have utilized this technique to gather additional evidence in support of the interaction of the PF with the GQ-DNA. As anisotropy dictates the extent of rigidity offered by surrounding environment, a high anisotropy value concludes a strong association between PF and DNA. Almost 10-fold rise in steady state anisotropy value (Figure 2b) is observed in the presence of 13.5 μM GQDNA inferring that the rotational motion of PF molecules significantly restricted when it binds to GQ-DNA through stacking interaction. 11092



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Figure 2. (a) Binding isotherm plot. The solid line indicates the nonlinear least-square fit (by IGOR Pro software) of the experimental data points using eq 2. (b) Steady state anisotropy plot of PF (4.5 μM) in the presence of varying concentration of G-DNA/μM.

Figure 3. (a) Fluorescence lifetime decay profiles of PF in buffer (pH 6.8) and in the presence of different concentrations of GQ-DNA. (b) Time-resolved fluorescence anisotropy decay profiles of PF in buffer (pH 6.8) and in the presence of different concentration GQ-DNA. All the legends carry respective meanings.

3.2. Time-Resolved Studies. Fluorescence lifetime measurement is an excellent technique to explore the excited-state environment around the fluorophores45 and hence can contribute significantly toward understanding binding interaction between PF and GQ-DNA. In PBS buffer (pH 6.8) PF exhibits a single exponential decay having a lifetime component around 4.8 ns (Figure 3, Table 1), which is in close agreement to previous reports.34 With the gradual increase of GQ-DNA concentration, a new short lifetime component of ∼800 ps starts appearing in the decay profile along with the 4.8 ns component (Table 1, Figure 3a). Moreover, the percentage of the reduced lifetime increases with concentration of GQ-DNA in the medium. We attribute this reduced lifetime component as an outcome of quenching effect by nucleobases, whereas the increased percentage of reduced lifetime is an indication toward enhanced extent of complexation between PF and DNA at higher concentration of GQ-DNA. The electron transfer from nucleobases to PF is responsible for the reduced lifetime of PF in the presence of GQ-DNA. This infers that PF involves in external stacking interaction at one or both the ends of Gquartet in order to take part in this electron transfer process. In summary, lifetime results are corroborative with our steady state results and predict that PF involves in π-type stacking interaction with the G-quartet. However, at this stage it will be

Table 1. Time-Resolved Fluorescence Decay Parameters of PF in the Absence and Presence of GQ-DNA, Collected at 510 nm sample PF (4.5 μM) in buffer PF(4.5 μM) + GQ (2.5 μM) PF (4.5 μM) + GQ (13.5 μM) a

τ1 (ns)

τ2 (ns)

a1

a2

τava (ns)

χ2

4.80 4.82

0.732

1 0.71

0.29

4.8 3.63

1.04 1.01

5.1

0.841

0.56

0.44

3.22

1.09

τav = a1τ1 + a2τ2

difficult to get a clear insight into the molecular picture of the interaction between PF and GQ-DNA. Excited state anisotropy measurements provide a notion about the rotational motion of the molecule, which directly reflects the extent of restriction imposed by the association process. Therefore, the time-resolved anisotropy measurement is employed to gather additional evidence in support of the interaction of PF with GQ-DNA. Time-resolved fluorescence anisotropy is measured using the following equation45 11093


The Journal of Physical Chemistry B r (t ) =

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which are characteristics features of antiparallel and parallel DNA, respectively (Figure 4a).43 The above-mentioned feature

I (t ) − GI⊥(t ) I (t ) + 2GI⊥(t )

(3)

where I∥(t) and I⊥(t) are fluorescence decays polarized parallel and perpendicular to the polarization of the excitation light, respectively. G is the correction factor for detector sensitivity to the polarization direction of the emission. Time-resolved anisotropy results are presented in Figure 3b, and results are summarized in Table 2. When the probe binds to a GQ-DNA, Table 2. Time-Resolved Anisotropy Decay Parameters of PF in the Absence and Presence of GQ-DNA, Collected at 510 nm sample PF (4.5 μM) blank. PF(4.5 μM) + GQ (2.5 μM) PF(4.5 μM) + GQ (13.5 μM) a

τr1 (ns)

τr2 (ns)

b1

b2

τra (ns)

χ2

0.175 0.245

2.55

1 0.77

0.23

0.175 0.774

1.07 1.01

0.360

3.39

0.29

0.71

2.525

1.08

τr = b1τr1 + b2τr2.

the rotational motion of the probe is expected to be retarded and is reflected through soaring rotational relaxation time of the probe. In the absence of GQ-DNA, the single exponential anisotropy decay is observed with rotational correlation time 175 ps. A single rotational decay time indicates that the probe molecules experience a homogeneous environment in aqueous buffer medium. In the presence of GQ-DNA, the anisotropy decay is found to be biexponential, which might be an outcome of distinctly different rotational relaxation times from free and DNA-bound PF molecules. In the presence of GQ-DNA a retarded component of 3.4 ns appears in the anisotropy decay profile along with the ∼175 ps component. The fast anisotropy component represents the rotational time of unbound PF, whereas the long component represents the rotational motion of DNA bound PF. The estimated rotational relaxation times (τr) are used to determine the hydrodynamic volumes of free and GQ-DNA bound PF molecules by using the following Stokes−Einstein relationship.46

τr =

ηV 1 = 6Dr kT

Figure 4. (a) Circular dichroism (CD) spectra for (3 + 1) hybrid GQDNA (5 μM) at different concentration of PF, where legends carry the respective meaning. (b) CD spectra depict PF-induced quadruplex formation in absence of any ions and induced CD appeared at 460 nm due to binding PF to GQ-DNA.

(4)

where Dr and η are the rotational diffusion coefficient and viscosity of the medium, respectively, and V is the hydrodynamic molecular volume of the complex at absolute temperature T. It is interesting to note that where free PF shows a hydrodynamic radius of 5.6 Å, the DNA bound PF shows a radius of ∼15 Å. The increased hydrodynamic radius is a further confirmation for the formation of association complex between GQ-DNA and PF. 3.3. Circular Dichroism (CD) and Thermal Melting (TM) Measurements: a Structural Glimpse of PF, GQDNA Noncovalent Interaction. Circular dichroism (CD) is a very sensitive technique to explore the modification of the secondary structure of biopolymers as a result of interaction with small molecules.47,48 Therefore, we have exploited the CD technique in order to examine PF induced structural alteration of GQ-DNA. In this study, GQ-DNA concentration is kept constant at 5 μM and changes of CD spectra are monitored with increasing concentrations of drug (PF). The CD spectra of GQ-DNA exhibits peak at ∼288 nm and shoulder at ∼270,

confirmed the hybrid (3 + 1) GQ-DNA (in which three strands are oriented in one direction and the fourth is in the opposite direction) structure formation and is consistent with previously reported literature for similar sequences of GQ-DNA in the presence of K+.43,49 Enhancement in CD signal with the gradual addition of PF indicates the slight perturbation of G-quadruplex structure in the presence of the ligand. Moreover, the CD signal at ∼288 nm shifts toward the shorter wavelength and the shoulder at ∼270 nm becomes more prominent in the presence of PF. The CD signal infers that the PF binding to GQ-DNA leads toward more parallel G-quadruplex structure, as the 270 nm peak is a characteristic feature of a parallel GQ-DNA.43 This kind of conformational switch has been reported previously wherein DNA binding ligands were shown to direct the folding of GQ-DNA into an alternate structure.43,48−50 Therefore, our results are also supportive toward the hypothesis about conformational switch that is induced by a DNA binding 11094



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molar ratio of PF to GQ-DNA in the lower panels. A standard nonlinear least-squares regression binding model for one-site binding is employed to fit the data. The best fit is shown in the lower panel. Thermodynamic parameters extracted from the fitting of binding curves are summarized in Table 3. From this study it is revealed that the binding is an enthalpically as well as entropically driven phenomenon with negative change in free energy (ΔG = −8.24 kcal/mol) having the binding affinity (Ka) of 1.13 × 106 M−1. The favorable enthalpy coming out from the PF and GQ-DNA association may be attributed to the base stacking as well as hydrogen bonding interactions between PF and nucleobases. Although the drug entropy is reduced significantly when PF binds to GQ-DNA, there are several other origins which results an overall positive entropy change during PF association with GQ-DNA. The loops present in the GQ-DNA have a considerable amount of hydration.39 Our CD results suggest that loop conformations are getting perturbed with the association of PF to the GQ-DNA. Thus, water molecules coming out from the loops during the binding process can increase system’s entropy. It is also reported that the water molecules participate to form a water network that lies along the grooves of GQ-DNA, making hydrogen-bonded contact with base edges and phosphate groups.39 There is a possibility that the binding of PF to GQ-DNA affects those hydrogen bond interactions, and thereby, water molecules coming out during the binding process increase the system’s entropy. The increased entropy may also appear due to an increase in mobility of the adenine residues in the edgewise loops. It was found earlier that when planner aromatic compounds interact with (3 + 1) hybrid G-quadruplex by end stacking it results in an increased mobility of adenine residues in edgewise loops.54,55 The PF binding induces destacking of these bases (A15 and A21) leading to increase in the configurational entropy of GQ-DNA. Interestingly, the number of binding sites of GQ-DNA determined from ITC are found to be nearly 1.5, which further supports the binding stoichiomety (GQ-DNA:PF = 2:3) determined from the break point in the Job’s plot56 (using fluorescence intensities at different molar ratios of PF (Figure 6b)). Previously, although several reports highlighted the interactions between different derivatives of PF and GQ-DNA,29−32 the literature lacks binding interaction between the precursor of those derivatives, i.e., PF and GQ-DNA. Therefore, it is worth to compare our results of binding interaction between PF and GQ-DNA with that of different derivatives of PF/acridine and GQ-DNA reported by several groups.29−32 Read et al.29 synthesized substituted acridine derivatives with increasing alkyl chain length and characterized the interaction with telomeric GQ-DNA through ITC and molecular modeling studies. They found that the binding constant decreases with increasing alkyl chain length of the substitution. In comparison to the maximum binding constant (9.72 × 104 M−1) obtained by them, we have observed ∼10-fold higher binding constant. Moreover, they observed much lesser change in enthalpy value compared to us. The results infer that increasing steric hindrance with higher length of alkyl substitution restricts the interaction with GQ-DNA. Very recently, Percivalle et al.30 reported interaction of synthetic acridine derivative with c-myc (Ka = 1.5 × 106 M−1) and c-kit2 (Ka = 3.6 × 105 M−1) GQDNA. Interestingly, the binding affinities observed by them are very close to our results. The most effective binding (Ka = 3.1 × 107 M−1) is observed for 3,6,9-trisubstituted acridine ligand (BRACO-19) and elucidated that ninth position anilino

ligand. Interestingly, an induced CD signal at around 460 nm further confirms binding of PF with GQ-DNA (Figure 4b). Since PF does not have any chirality, no CD signal can be expected for free PF. However, GQ-DNA bound PF shows pronounced induced CD signal near 460 nm (Figure 4b) inferring that PF involves base stacking interaction with GQDNA. Another important finding observed in the CD study is that PF induces the formation of GQ-DNA from single stranded human telomeric DNA (H24) even in the absence of any K+ ions. It is evident from Figure 4b that a new peak, which appeared at ∼270 upon addition of PF, is a signature for parallel G-quadruplex DNA. This infers that PF also provides stability to the GQ-DNA like other ligands such as ellipticine,44 cresyl violet,51 and thioflavin-T.50 To confirm whether PF binding to GQ-DNA leads to any structural stabilization or not, we have carried out thermal melting study for free and PF bound GQ-DNA. Thermal melting profiles of GQ-DNA and PF bound GQ-DNA are shown in Figure 5. Binding of PF to GQ-DNA offers an ∼8 °C

Figure 5. Melting curve for GQ-DNA (2.2 μM) in the absence and presence of PF (0.7 μM) collected at 290 nm.

(melting temperature of GQ-DNA alone is 63.5 °C, whereas melting temperature of GQ-DNA in the presence of PF is 71.4 °C) increase in melting temperature, which infers structural stabilization of GQ-DNA due to PF binding. It has been earlier reported that ethidium derivatives binding to GQ-DNA (increase in melting temperature between 7 to 10 °C) inhibits the telomerase activity with high affinity (IC50 in nM).52 Therefore, considering an 8 °C increase in melting temperature in the case of PF bound GQ-DNA, it is reasonable to assume that PF might also act as a potential telomerase inhibitor. 3.4. Isothermal Titration Calorimetry (ITC) Study, Thermodynamic Glimpse of Binding. To obtain thermodynamic parameters of PF binding with GQ-DNA, isothermal titration calorimetry (ITC) experiment is performed. It is an effective and sensitive tool for characterizing small molecular binding to biomacromolecules and might provide key insights about the molecular forces responsible for complex formation, number of binding sites, and binding energy.53 Isothermal titration calorimetric profiles for PF with GQ-DNA are shown in Figure 6a. Each peak of the binding isotherm in the upper panels represents each of the PF injections. The amount of heat liberated by successive addition of PF is plotted against the 11095



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Figure 6. (a) Upper panel shows the isothermal titration plot of GQ-DNA (in cell) with PF (in syringe), whereas the lower panel shows the integrated heat profile of the calorimetric titration plot shown in upper panel. The solid line represents the best nonlinear least-squares fit to a single binding site model. (b) Job’s plot for the association of PF with GQ-DNA. Here χPF represents mole fraction of PF.

Table 3. Thermodynamic Parameters from Isothermal Titration Calorimetry Experiments binding constant (Ka) (mol−1)

N

ΔH (kcal mol−1)

ΔS (cal mol−1 K−1)

ΔG (kcal mol−1)

(1.13 ± 0.65) × 10

1.37 ± 0.18

−2.042 ± 0.37

20.8

−8.24

6

substitution in acridine derivative plays a key role to enhance the quadruplex affinity.32 Moreover, using the crystal structure they proposed that 3-,6- substituents (comprising flexible side chains with a terminal five-membered pyrrolidino ring) bind to the quadruplex groove and offer an enhanced stability to the complex.32 Therefore, the observed less binding affinity for PF compared to some of its synthetic derivatives may be attributed to the lack of side chain substituent, which stabilizes the GQDNA through groove binding interaction. 3.5. Competitive Replacement Assay Experiment. To confirm the location of drug on GQ-DNA, we have performed competitive replacement assay experiment using ethidium bromide (EB), which emits fluorescence at ∼605 nm. In this experiment, mainly steady state anisotropy of PF at 510 nm is monitored. It is evident from Figure 7 that the steady state anisotropy value of PF increases from 0.025 to 0.132 with addition of GQ-DNA, confirming that PF molecules bind to GQ-DNA. When EB is added to this PF-bound GQ-DNA solution, the anisotropy value decreases with increase in concentration of EB. After a certain concentration of EB (2.5 μM), the anisotropy is found to be constant at 0.077 which is in between free PF (0.025) and GQ-DNA bound PF value (0.132). Based on the above observations, we envisage that few PF molecules bound to GQ-DNA are replaced by EtBr, as it has already been reported that EtBr binds to GQ-DNA through terminal end stacking mode.52 The above observation also infers that PF molecules, those are stacked at terminal ends, are replaced by EtBr, whereas PF molecule sandwiched in between two GQ-DNA remains bound. As a result, the steady state anisotropy value remains constant after certain addition of EB.

Figure 7. Steady state anisotropy of PF monitored at 510 nm in the presence of GQ-DNA and EtBr.

If EB would have replaced all the PF molecules, then the anisotropy value should have reverted to its original value. Therefore, the above results support our conjecture that one PF molecule is sandwiched between two GQ-DNA and other two PF molecules are bound (end stacked) to the two remaining Gtetrads of GQ-DNA (Scheme 2). 3.6. Molecular Picture of Interaction between PF and GQ-DNA. Literature reports of binding stoichiometries for the small molecules interacting with G-quadruplex are highly diverse. Planar aromatic compounds are known to bind with 11096



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which inhibits the replication process of the telomeric sequence. The observed binding mode for PF can be more effective for the inhibition process, as in this 2:3 binding mode it blocks both of the ends of the quadruplex and, thereby, hampers the interaction with telomerase, a replicase enzyme. Moreover, a higher stoichiometry of binding interaction enhances the thermodynamic stability of the quadruplex structure, which is reflected by the 8 °C increase in melting temperature of PF bound GQ-DNA as well as the higher favorable free energy change for the binding process. Therefore, based on the results we propose that, along with DNA intercalation, interaction with telomeric DNA region and the subsequent retardation of telomerase activity might be an extra mode of action for PF anticancer property.

Scheme 2. Proposed Molecular Picture of Binding Interaction between PF and (3 + 1) Hybrid Human Telomeric GQ-DNA

4. CONCLUSION In this work, we have studied the interaction of PF (a wellknown DNA intercalator, hence acting as anticarcinogen) with a human telomeric DNA sequence, which forms a hybrid (in which three strands are oriented in one direction and the fourth is in the opposite direction) G-quadruplex DNA (GQ-DNA) structure in the presence of K+ ion. Spectroscopic and calorimetric studies indicate that the association of PF with GQ-DNA is an enthalpically as well as entropically driven phenomenon with an intrinsic association constants of 1.15 × 106 M−1. Interestingly, PF binding induces GQ-DNA more toward a parallel structure and provides extra stabilization to GQ-DNA, which is reflected from the rise in melting temperature of ∼8 °C. With the observed binding stoichiometry (PF:GQ-DNA = 3:2) from isothermal titration calorimetry (ITC) and Job’s plot, we propose that one molecule of PF gets sandwiched between two GQ-DNA and two PF molecules that are bound (end stacked) to each of the two remaining Gtetrads of GQ-DNA.

GQ-DNA via external end π−π stacking to the surface of the Gquartet at the one or both of ends.18,54,55 It is interesting to note that several quadruplex ligands (e.g., porphyrins,57 plant alkaloids (berberine and sanguinarine),58,59 perylene derivatives,60 ellipticine,44 telomestatin,61 macrocyclic oligoamide,62 and acridine derivatives29−32) have been reported to bind to telomeric G-quadruplexes in the end-stacking mode with higher binding stoichiometry. Combining all these previous reports along with the observed binding stoichiometry (PF:GQ-DNA = 3:2) from ITC and Job’s plot, we propose that each GQ DNA binds to 1.5 molecules of PF. This observation prompted us to conclude that one molecule of PF gets sandwiched between two molecules of GQ-DNA and other two PF molecules are bound (end stacked) to the two remaining G-tetrads of GQDNA (Scheme 2). As we have not found any evidence for the concentration dependency complex formation in our study, it is reasonable to assume the PF-GQ-DNA complex will form a head to head or tail to tail interaction but not head to tail interaction. Notably, ellipticine, another potent anticancer drug, also exhibited similar kinds of stoichiometry, orientation and binding constant.44 Like PF, ellipticine is also a well-known DNA intercalator having an almost planar structure. Moreover, both ellipticine and PF contain positive charge and both of them do not contain any side chain long substituent. Based on our results as well as molecular picture of binding interaction of acridine derivatives provided by several groups,29−32 we infer that PF orients on the quadruplex plane by keeping the protonated N atom of the ring toward the K+ ion channel of the GQ-DNA. Moreover, due to its extended π clouds, PF participates in strong π−π stacking interactions mainly with guanine nucleobases, which causes PF to bind at both the ends of G-quartets (in between the two side thymine loops) in an intercalation-type arrangement reminiscent of intercalation into duplex DNA. Moreover, association with PF probably causes destacking of the two adenine bases (A15 and A21) situated at both the ends of G-tetrads (Scheme 2). Conventionally, PF is a well-known intercalator for duplex DNA, and in this mode of interaction PF intercalates between the base pairs and gets stabilization through stacking interactions with the nucleobases. This intercalation process elongates the DNA and makes it nondetectable for DNA replicase enzyme and thereby prevents the DNA replication process. The tumor cell proliferation can also be regulated more effectively by the structural stabilization of telomeric GQDNA (detail has been discussed already in the Introduction),

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +91-20-2590-8077. Fax: +91-20-2589 9790. Author Contributions †

These authors (V.K. and A.S.) contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS P.H. is thankful to Council for Scientific and Industrial Research (CSIR) (Scheme no. 37(1499)/11/EMR-II) for financial support. V.K., A.S., and R.K.K. are thankful to Department of Science and Technology (DST), CSIR and University Grants Commission (UGC) for the INSPIRE fellowship, Senior Research Fellowship (SRF) and Junior Research Fellowship (JRF), respectively. The authors are indebted to Director, IISER-Pune for providing excellent experimental facilities. The authors thank Dr. H. N. Gopi for his valuable suggestions related to ITC experiments and results. The authors highly appreciate the active participation of Mr. Sagar Satpathi for performing certain spectroscopic experiments. The authors are thankful to an editor and anonymous reviewers for their valuable comments and suggestions. The authors thank Dr. Arnab Mukherjee for helping us with nonlinear fitting analysis. 11097



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