Spectroscopic and viscometric elucidation of the interaction between a potential chloride channel blocker and calf-thymus DNA: the effect of medium ionic strength on the binding mode

Abstract

The present study demonstrates a detailed characterization of the binding interaction of a potential chloride channel blocker 9-methyl anthroate (9-MA) with calf-thymus DNA. The modulated photophysical properties of the emissive molecule within the microheterogeneous bio-assembly have been spectroscopically exploited to monitor the drug–DNA binding interaction. Experimental results based on fluorescence and absorption spectroscopy aided with DNA-melting, viscometric and circular dichroism studies unambiguously establish the binding mode between the drug and DNA to be principally intercalative. Concomitantly, a discernible dependence of the mode of binding between the concerned moieties on the ionic strength of the medium is noteworthy. A dip-and-rise characteristic of the rotational relaxation profile of the drug within the DNA environment has been argued to be originating from a substantial difference in the lifetime as well as amplitude of the free and DNA bound drug molecule. In view of the prospective biological applications of the drug, the issue of facile dissociation of the intercalated drug from the DNA helix via a simple detergent-sequestration technique has also been unveiled. The utility of the present work resides in exploring the potential applicability of the fluorescence properties of 9-MA for studying its interactions with other relevant biological or biomimicking targets.

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Introduction

Developing a clear understanding of the interaction of small molecules with various biological targets has formed the nucleus of many-faceted research activities for the perception of structural and functional features of biomolecules so as to simulate the biophysical processes.¹ Of them, deoxyribonucleic acid (DNA) is often considered as the most essential target as it contains the instructions for the development and functioning of living organisms.^2,3 Spectroscopic portrayal of the interaction of small molecules with DNA not only provides biological insights, but also furnishes opportunity towards the development of new efficient therapeutic agents to control gene expression.^3–5 Such interaction in general involves two dominant binding modes referred to as (i) intercalative binding where the molecule fits within the nucleic acid base pairs distorting the DNA backbone and (ii) groove binding involving non-bonding interaction with the deep major or the shallow minor groove of the helix, the former being the most effective mode for drugs targeted to DNA.^4–6 The affinity, strength and mode of such binding interactions are governed by a variety of structural and electronic factors. Although so far no specificity has been clearly imposed on the structure of the small molecule to predict its mode of binding with DNA, it is a well speculated fact that most of the planar aromatic molecules tend to bind to DNA essentially in an intercalative fashion whereas crescent shaped molecules in general prefer the groove binding mode.^5–7 For polar or cationic molecules, a third mode of communication viz. electrostatic interaction with the negatively charged DNA backbone becomes conspicuous and it is also anticipated that such interaction can efficiently modulate the strength of intercalation or groove binding.^8,9

Variation of ionic strength of the medium is known to have a marked influence on the drug–DNA binding phenomenon. At a high ionic strength, the highly charged DNA backbone tends to stabilize due to charge screening and consequently, the electrostatic contribution towards the binding event diminishes, inducing a noteworthy drop in the binding energy.^10,11 Although no report of a severe conformational change of the macromolecule with the increasing ionic strength of the medium is available to date, it is a known, though not so well nurtured, fact that a change in the ionic strength of the solution could result in an alteration of the binding mode of the drug with DNA.^12,13 While a low ionic strength favors intercalation, at a high ionic strength the groove binding mode becomes the governing one.^12–14

The present contribution thus describes the binding interaction of 9-methyl anthroate (9-MA, videScheme 1), a molecule belonging to the eminent chloride channel blocker family as well as known to inhibit fatty acid incorporation into phospholipids in human airway epithelial cells,¹⁵ with calf-thymus DNA with the varying ionic strength of the medium. The modulated excited state photophysics of the drug within the microheterogeneous bio-environment has been used as the actuating tool to monitor the binding process, whereas the changes in the conformation of the biopolymer exerted by the drug have been explored using a circular dichroism study. Moreover, with a view towards the prominent physiological activities of the studied molecule, we have attempted to search for a simple strategy for the dissociation of the drug from the drug–DNA complex. Our results do not only indicate that the straightforward strategy of detergent-sequestration¹⁶ can be effectively used to achieve the goal but a slower rate of dissociation also substantiates the claim of the prospective use of the drug as a therapeutic agent.

Scheme 1 Structure of 9-methyl anthroate (9-MA).

Experimental

Materials

The detailed synthesis and purification procedure of 9-MA (videScheme 1) has been described elsewhere.¹⁷ Calf-thymus DNA from Sigma-Aldrich, USA, was used as received. Tris buffer was purchased from SRL, India, and 0.01 M Tris-HCl buffer of pH 7.4 was prepared in triply distilled deionized water from a Milli-Q water purification system (Millipore). The solvent appeared visually transparent and the purity was also tested by running the fluorescence spectra in the studied wavelength range. Acrylamide, guanidine hydrochloride (GuHCl), sodium chloride (NaCl) and cetyl-trimethyl ammonium bromide (CTAB) were purchased from E-Merck and used as received. Stock solution of DNA was prepared by dissolving solid DNA in Tris buffer followed by thorough sonication and repeated filtration. The final solution was stored at 4 °C. The purity of the DNA sample was verified by monitoring the ratio of absorbance at 260 nm to that at 280 nm which was within the range of 1.8–1.9.⁴ The concentration of DNA in the solution was determined spectrophotometrically using the molar absorption coefficient ε_DNA = 12 858 cm⁻¹ M⁻¹ at 260 nm.¹⁸ In all the spectral measurements, the concentration of the drug was maintained at ∼2.0 μM to avoid aggregation and re-absorption effects.

Instrumentation and methods

Steady-state spectral measurements. The absorption and emission spectra were recorded on a Hitachi UV-Vis U-3501 spectrophotometer and Perkin-Elmer LS55 fluorimeter, respectively, with appropriate corrections for instrumental response. The recorded spectra were appropriately background subtracted by a similar set of solutions under experiment with the drug being omitted, in order to eliminate any spectral interference. Experiments have been carried out at an ambient temperature of 298 K, unless otherwise specified. Only freshly prepared solutions were used for spectroscopic measurements. For the helix melting experiment, the prefixed temperatures were set by circulating water from a Neslab RTE-100 (USA, temperature stability within ±0.01 °C) water bath attached to the absorption spectrophotometer. The association and dissociation kinetics of the drug with DNA have been measured on the aforementioned fluorimeter using the stopped-flow fluorescence measurement technique. The dead-time of the instrument was found to be ∼20 ms.

Time-resolved fluorescence decay measurements. Fluorescence lifetimes were obtained by the method of Time Correlated Single-Photon Counting (TCSPC) on a FluoroCube-01-NL spectrometer (Horiba Jobin Yvon) using a Laser-Diode having an output of 375 nm and the signals were collected at the magic angle of 54.7° to eliminate any considerable contribution from fluorescence anisotropy decay.¹⁶ The typical time resolution of our setup is ∼100 ps. The decays were deconvoluted on DAS-6 decay analysis software. The acceptability of the fits was judged by χ² criteria and visual inspection of the residuals of the fitted function to the data. Average lifetimes were calculated using the standard procedure.¹⁷

For time-resolved fluorescence anisotropy decay measurements, the polarized fluorescence decays for the parallel [I_VV] and perpendicular [I_VH] emission polarizations with respect to the vertical excitation polarization were first collected at the emission maxima of the drug. The anisotropy decay function r(t) was constructed from these I_VV and I_VH decays using the standard procedure.¹⁹

Viscometric study. Viscosity measurements were made using an Ubbelohde viscometer that was maintained at 298 K using a constant temperature bath. The volumes of the solutions were fixed at 8.0 mL, and flow time was measured using a digital stopwatch. To estimate the viscosity of the sample, the mean of three replicated measurements was taken.

Circular dichroism spectroscopy. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter using a cylindrical cuvette of 0.1 cm path-length at 25 °C. The reported CD profiles are an average of four successive scans obtained at 20 nm min⁻¹ scan rate with an appropriately corrected baseline. The concentration of DNA and the drug during CD measurements are mentioned in the relevant discussion.

Results and discussion

Steady state absorption and emission spectral study

The absorption profile of 9-MA is characterized by a broad structured band with a maximum at ∼360 nm, attributed to the S₁ ← S₀ transition (π–π* type) of the anthracene chromophore. The emission profile of the drug consists of a large Stokes shifted, unstructured band, which being polarity sensitive, exhibits a substantial red shift of ∼45 nm upon moving from n-hexane to water,¹⁷ thereby signifying its efficacy as an external probe to study biological and biomimicking microenvironments.

Upon the addition of DNA to the solution of 9-MA in aqueous buffer, the absorption spectra of the drug shows a steady decrement in absorbance along with a red shift of the absorption maximum (videFig. 1a), indicating a lesser exposure of the drug towards the bulk medium as well as a significant modification of the microenvironment surrounding the drug suggesting a binding interaction. However, it is worth noting in the present context that the DNA-induced absorption spectral changes of 9-MA lack an isosbestic point (inset of Fig. 1a), indicating the operation of more than one type of interactions during the binding process.²⁰ Hence, we have not employed the absorption study for the determination of the binding constant.

Fig. 1 (a) Absorption spectra and (b) emission spectra (λ_ex = 360 nm) of 9-MA in the presence of increasing concentration of DNA. Curves i → vi (vide (a)) correspond to [DNA] = 0, 50, 100, 200, 400, and 700 μM, and curves i → viii (vide (b)) correspond to [DNA] = 0, 50, 75, 100, 200, 300, 500 and 700 μM.

The emission profile of the fluorophore was found to reflect the results of the drug–DNA interaction more vividly. The emission maximum of the drug is observed at ∼485 nm in aqueous buffer medium. With increasing concentration of DNA, a prominent enhancement of the emission intensity along with a distinct blue shift of the emission maxima (from λ_em ∼ 485 nm in aqueous medium to ∼467 nm in 750 μM DNA, videFig. 1b) is observed. Such observations confirm that the polarity of the modified microenvironment around the drug is more hydrophobic compared to the bulk aqueous phase.

In the present context, we have also made an endeavor to estimate the polarity of the immediate microenvironment around the fluorophore. For this, we have employed the sensitivity of its emission band towards the polarity of the medium, by taking the drug in water–dioxane mixtures of varying compositions having precisely known polarity in terms of the standard E_T(30) scale²¹ and comparing those with the spectral properties of the fluorophore in the concerned microenvironment. A calibration plot monitoring the energies corresponding to the emission maximum of 9-MA in the water–dioxane mixtures of known E_T(30) has been exploited for this purpose. The micropolarity of the binding region is thus determined to be 54.39 on a E_T(30) scale, which is considerably low compared to that of bulk water (E_T(30) = 63.1) indicating the significant hydrophobic character of the binding site.²² However, considering the possibilities of specific solvation of the drug in this solvent mixture as well as the gross approximation that these binary solvents mimic the real bio-environment, the E_T(30) value thus obtained should be assessed as only a qualitative one.

Since the estimation of the binding constant can furnish some idea about the mode of binding, we have examined the emission data using the modified Benesi–Hildebrand equation,²³ as follows,

in which I₀, I and I₁ are the emission intensities, respectively, in the absence, at intermediate and infinite concentration (indicating saturation of interaction) of DNA, whereas K_a is the association constant. The plot of 1/[I – I₀] vs. 1/[DNA] produces a straight line (videFig. 2a) justifying a 1 : 1 interaction between the drug and DNA.²³ The corresponding binding constant (K_a) is determined from the intercept to slope ratio of the aforesaid plot and the computed value is K_a = (5.84 ± 0.4) × 10⁴ M⁻¹ at 298 K. The order of the evaluated binding constant indicates a moderately strong binding of the drug with DNA. Using this value of K_a, the free energy change for this process is determined to be ΔG = −26.99 kJ mol⁻¹, which dictates a favorable complexation process.

Fig. 2 (a) Benesi–Hildebrand plot of 1/[I − I₀] vs. 100/[DNA] (μM⁻¹) for binding of 9-MA with DNA. (b) Stern–Volmer plots for fluorescence quenching of 9-MA by acrylamide under various conditions as indicated in the figure legends. Each data point is an average of 5 individual measurements. The error bars are within the marker symbols if not apparent. (c) Variation of steady-state fluorescence anisotropy (r) of the drug as a function of DNA concentration. Inset shows the variation of steady-state fluorescence anisotropy of 9-MA as a function of composition of glycerol–water mixtures. Each data point is an average of 15 individual measurements. The error bars are within the marker symbols if not apparent.

To elucidate the mode of binding, fluorescence quenching of DNA bound 9-MA is studied in relation to the fluorescence quenching of the drug in buffered aqueous solution. We have used neutral acrylamide as the quencher to avoid the complicacies associated with the interaction between the ionic quenchers and the DNA itself. Considering the two different possible locations of the entrapped drug, i.e., into the DNA double strands for intercalative binding and the deep major or shallow minor grooves for groove binding, the protection of the fluorophore from the quencher is expected to be more in the former case and less for the latter situation.^20,22 The fluorescence quenching of 9-MA with the addition of the quencher has been followed using the archetype Stern–Volmer equation,^20,22i.e.

in which I₀ is the original fluorescence intensity; I is the quenched intensity of the fluorophore (9-MA); Q is the quencher (here acrylamide); and K_SV is the Stern–Volmer quenching constant. Fig. 2b represents the Stern–Volmer plot and depicts the relative extent of quenching. Acrylamide efficiently quenches the fluorescence of 9-MA corresponding to K_SV = (29.44 ± 0.55) M⁻¹. However, when the same quenching experiment was performed on the DNA-bound drug molecule ([DNA] = 1 mM, this concentration is chosen to ensure the saturation of interaction between the drug and DNA), the extent of quenching is found to be drastically reduced with corresponding K_SV = (8.39 ± 0.2) M⁻¹. Since acrylamide does not bind to DNA which might lead to a reduced efficacy of the molecule to serve as a quencher,²⁴ this result at once supports an intercalative mode of binding of 9-MA into the DNA double strands.

Steady state emission anisotropy study

The microenvironment associated with the drug molecule is governed by its precise location in a complex molecular assembly.^20,22 Any modulation in the rigidity of the surrounding atmosphere of the fluorophore is thoroughly manifested through anisotropy variation. In the present scenario, the fluorescence anisotropy of 9-MA (videFig. 2c) exhibits a specific variation as a function of the DNA concentration. An initial steep rise in the anisotropy value implies the increasing degree of motional restriction on the drug molecules upon binding to DNA, which is then followed by a gradual saturation.

We have extended the anisotropy measurements of the drug 9-MA in glycerol–water mixture of different compositions and compared the values with the anisotropy values of the DNA bound situations (vide the inset of Fig. 2c) to get a quantitative insight into the motional restriction imposed on the DNA-bound drug. We have noticed that the anisotropy value of the drug in 1 : 1 glycerol–water mixture is almost similar to that in the DNA environment at the saturation level which signifies a high enough motional rigidity imposed on the drug and is thus suggestive of intercalation. Again owing to the presence of oxygen atoms in the drug molecule which are excellent hydrogen-bond acceptors, the possibility of specific interaction can not be ruled out.

Confirming the mode of complexation: thermal melting and viscometric study

DNA melting is the process by which the non-bonding interactions between the strands of the double helix are broken by an external stimulus (such as temperature) leading to a separation of the two. The melting temperature (T_m) of DNA is defined as the temperature at which half of the DNA strands are in the double-helical state and the other half are in the random-coil state.^20,22 Intercalation of small molecules to DNA is known to enhance the thermal stability of the helix due to stabilizing π–π stacking interactions (whereas electrostatic or groove-binding leads to only a minute modulation), which gets manifested via an increment of the melting temperature. Therefore, any significant perturbation in the T_m of the DNA helix in the presence of the drug can provide a convincing evidence for the intercalative mode of binding.^20,22 The DNA melting profile (in the absence and presence of the drug 9-MA) has been constructed by monitoring the absorbance of DNA at 260 nm, which displays a hyperchromic shift with increasing temperature which is regarded as the signature of helix melting.^20,22Fig. 3a clearly reflects the ability of 9-MA to improve the thermal stability of DNA (shifting of T_m from ∼78.8 °C in the absence of 9-MA to ∼84 °C in the presence of 9-MA), which strongly supports the intercalative mode of binding of 9-MA into the DNA helix.

Fig. 3 (a) Thermal melting profiles of DNA and the 9-MA–DNA complex as constructed by observing the relative absorbance at λ_abs = 260 nm as a function of temperature. (b) Effect of increasing amounts of EtBr and 9-MA on the relative specific viscosity of DNA (concentration of DNA was maintained at ∼50 μM). Each data point is an average of 5 individual measurements. The error bars are within the marker symbols if not apparent.

On the other hand, it is often argued that photophysical properties provide inadequate clues to support the binding mode of molecules with DNA whereas hydrodynamic studies are regarded more fruitful owing to their sensitivity towards length changes.^25,26 A classical intercalation model demands lengthening of the DNA helix as base pairs get separated to accommodate the binding molecule, leading to an increased viscosity of the solution.^25,26 In favor of this standpoint, the plot of relative specific viscosity against the [drug–DNA complex]/[DNA] ratio (Fig. 3b) illustrates a steady increase in viscosity with increasing drug concentration, and the rate of increment is qualitatively comparable with a classical DNA intercalator, ethidium bromide (EtBr, videFig. 3b), thereby suggesting that the drug intercalates into the base pairs causing an extension of the helix.^25,26

Chaotrope induced perturbation of the drug–DNA binding phenomenon

It is a well-established fact that the denaturation of DNA leads to a weakening of drug–DNA binding, which results in the release of the entrapped drug to the bulk. Here guanidium hydrochloride (GuHCl) induced modifications of the spectral characteristics of DNA-bound 9-MA have been followed by steady state fluorescence measurements to seek further confirmation in support of 9-MA–DNA binding. Gradual addition of GuHCl to the drug–DNA complex modifies the fluorescence spectra of the drug in a manner qualitatively opposite to that observed in the case of gradual addition of DNA to the aqueous solution of the drug. The fluorescence intensity exhibits a progressive reduction with increasing GuHCl concentration, with a simultaneous shift of the emission maximum to the red (figure not shown), indicating the enhanced exposure of the drug molecule towards the bulk phase. It is also stimulating to note that at a substantial concentration of GuHCl (8 M), the emission wavelength of 9-MA tend to correspond to the values in aqueous buffer solution (from ∼467 nm in 0 M GuHCl to ∼482 nm in 8 M GuHCl). These observations are in line with the idea that the denaturant is able to release the fluorophore completely from the DNA environment to the bulk aqueous phase.

This idea of the release of the drug to the bulk aqueous phase in the presence of the denaturant is further corroborated by a thorough inspection of the steady-state fluorescence anisotropy values. It appears that despite the steady decrement of the anisotropy of DNA-bound drug with increasing chaotrope concentration further confirming the release of the drug to the bulk (figure not shown), the anisotropy value at a reasonable GuHCl concentration remains still somewhat higher than the value in the absence of DNA (r ∼ 0.04 for the free drug in aqueous buffer vs. r ∼ 0.065 for the drug in the presence of 8 M GuHCl). This observation is possibly the outcome of an increase in the bulk viscosity of the solution due to the addition of GuHCl.

Effect of the ionic strength of the solution on the binding phenomenon

Increased ionic strength of the solution neutralizes the negative charges of the phosphate backbone of the DNA, resulting in a shrinking of the helix due to a reduction in the unwinding tendency caused by the electrostatic repulsions present in between the phosphate groups.¹⁰ Such a collapse of the helix is expected to adversely affect both intercalation and groove binding. Fig. 4a reveals the effect of increasing ionic strength of the medium (via the addition of NaCl) on the emission properties of the DNA-bound drug. Increase in the ionic strength of the medium is found to initiate further fluorescence enhancement of the DNA-bound drug, without any noticeable shift of the emission maximum, which suggests further diminution of the non-radiative decay channels of the excited fluorophore, the reason behind which has been illustrated in the forthcoming sections.

Fig. 4 (a) Representative emission profile of DNA-bound 9-MA with increasing ionic strength of the medium. Curves i → viii correspond to [NaCl] (mM) = 0, 50, 100, 150, 200, 250, 300, 400 and 500 ([DNA] = 750 μM). (b) Representative time-resolved fluorescence decay profiles (λ_ex = 375 nm, λ_monitored = λ_max(em)) of the drug (9-MA) in the presence of increasing DNA concentrations. The sharp black profile on the extreme left represents the instrument response function (IRF). Curves i → vi correspond to [DNA] = 0, 50, 100, 250, 500 and 750 μM. (c) Representative time-resolved fluorescence decay profiles (λ_ex = 375 nm, λ_monitored = λ_max(em)) of the DNA-bound drug (9-MA) in the presence of increasing NaCl concentrations ([DNA] = 750 μM). Curves i → v correspond to [NaCl] = 0, 100, 200, 300 and 500 mM.

Time-resolved fluorescence and anisotropy decay measurements

In an attempt to follow a generalized picture of the interactions involved, we have chosen to record the fluorescence decays of 9-MA in a series of concentrations of DNA. Some representative time-resolved decay profiles are displayed in Fig. 4b and the relevant parameters are presented in Table 1. The drug molecule is found to exhibit a bi-exponential decay pattern in a bulk aqueous buffer phase,¹⁷ which comprises of a fast component (∼310 ps) ascribed to the free drug (having a contribution of ∼99.4%) and another slower component (3.14 ns) attributed to the solvated (hydrated) cluster of the drug (with a meager contribution of ∼0.6% only).

Table 1 Time-resolved fluorescence decay parameters of 9-MA with increasing concentrations of DNA

[DNA] in μM	α 1 (%)	α 2 (%)	α 3 (%)	τ 1 (ps)	τ 2 (ns)	τ 3 (ns)	〈τf〉 (ns)	χ ^2
a Standard deviation for the fitting analysis is = ±5%.
0	99.35	0.66	—	310	3.14	—	0.51	0.98
50	99.09	0.36	0.55	319	2.86	8.56	1.43	1.11
75	98.68	0.50	0.82	323	2.83	9.87	2.28	1.09
100	98.41	0.58	1.01	321	2.97	10.33	2.81	1.13
150	98.11	0.64	1.25	325	3.24	10.37	3.23	1.12
200	97.67	0.72	1.61	326	3.71	13.52	5.59	1.15
250	96.82	0.85	2.33	323	3.57	14.63	7.61	1.03
400	96.34	0.94	2.72	328	3.66	15.25	8.57	1.05
500	95.67	1.06	3.27	327	3.94	15.22	9.18	1.15
600	95.04	1.18	3.78	329	3.87	15.16	9.63	1.06
750	94.47	1.23	4.30	331	3.87	15.28	10.15	1.04

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As is obvious from the data assembled in Table 1, the time-resolved fluorescence decay of the DNA-bound drug can be satisfactorily described only by a complex tri-exponential pattern with three distinct lifetime components. The component τ₁ having the highest contribution (which shows no appreciable change in magnitude but a decrement in amplitude with increasing DNA concentration, videTable 1) is anticipated to correspond to the free (i.e. unbound) drug whereas, the third component τ₃, which shows a massive increment in magnitude coupled with a complementary increase in the amplitude with increasing DNA content (from ∼8.5 ns in 50 μM DNA to ∼15.3 ns in ∼750 μM DNA, videTable 1) is predicted to be the outcome of the intercalation of the drug within the DNA helix. The second component τ₂ having an intermediate lifetime (within the range ∼2.8–3.8 ns) is of special interest since its magnitude being significantly larger than that of the free drug in solution implies that the corresponding species is obviously an outcome of a binding phenomenon between the drug and DNA, the probable mode being non-intercalative. This component also corroborates to the absence of an isosbestic point in the absorption spectra indicating more than one mode of binding. We, thereby, connect it with the possibility of groove binding or some electrostatic interaction of the drug with the biomolecule. Moreover if we consider the mean (average) fluorescence lifetime of the drug only, it is observed that, the average lifetime (〈τ_f〉) of 9-MA progressively increases with increasing DNA concentration and the occurrence of a binding interaction is thus further confirmed.

A more critical analysis of the data has been undertaken with a view to demarcate the contributions from radiative (k_r) and non-radiative (k_nr) decay constants of the drug in aqueous buffer and DNA environments according to the following equations:²⁴

k_r + k_nr = 〈τ_f〉⁻¹ (4)

where Φ_f denotes fluorescence quantum yield of the drug. The calculated values are summarized in Table 2 which readily infer that in DNA medium the non-radiative decay constants k_nr are reasonably reduced from that in the aqueous buffer medium (whereas the radiative decay rates are only faintly disturbed). Thus, the improved lifetime of the drug in the DNA environment seems to be the outcome of the attenuation of radiationless decay paths via the motional restriction imposed on the drug.

Table 2 Fluorescence quantum yield (Φ_f) and kinetic fluorometric parameters for 9-MA with increasing concentrations of DNA

[DNA] in μM	〈τf〉 (ns)	Quantum yield (Φf)	k r (s^−1) × 10^−7	k nr (s^−1) × 10^−7
0	0.51	0.010	1.961	194.118
50	1.43	0.013	0.909	69.021
75	2.28	0.018	0.789	43.070
100	2.81	0.025	0.889	34.698
150	3.23	0.038	1.176	29.783
200	5.59	0.051	0.912	16.977
250	7.61	0.066	0.867	12.273
400	8.57	0.082	0.957	10.712
500	9.18	0.116	1.264	9.630
600	9.63	0.144	1.495	8.889
750	10.15	0.165	1.626	8.227

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In this regard, it is also pertinent to analyze the results obtained from the examination of the effect of ionic strength of the solution on the dynamics of the DNA-bound drug (videFig. 4c and Table 3). The data compiled in Table 3 shows that the corresponding decay profiles can also be satisfactorily deconvoluted using a tri-exponential fitting function as earlier and the average lifetime (〈τ_f〉) of the drug progressively decreases with increasing ionic strength of the medium. As expected, with increasing salt concentration the amplitude as well as the magnitude of the lifetime component corresponding to the intercalated drug (i.e. τ₃) is found to considerably decrease owing to the diminution of the electrostatic contribution towards the intercalation process. Interestingly, with increasing ionic strength of the medium, there is a discernible increase in the second lifetime component (i.e. τ₂) both in magnitude and amplitude (videTable 3). This observation rules out the possibility of assigning this lifetime component towards any process associated with electrostatic interaction, rather it indicates groove binding which is recognized to be favored at high ionic strength.¹⁴ A slight increase in the lifetime corresponding to the free drug (τ₁) could be the outcome of an increase in viscosity of the solution due to the presence of an appreciable concentration of salt. From the tabulated data, it is also worth noting that the population of the DNA-bound drug (via both intercalation and groove binding) progressively increases with increasing ionic strength of the medium (from ∼5.5% in the absence of NaCl to ∼14.5% in the presence of 500 mM NaCl). The above scenario thus casts light on the observed fluorescence enhancement of the drug with increasing ionic strength of the solution.

Table 3 Time-resolved fluorescence decay parameters of 9-MA with increasing concentrations of NaCl in the presence of 750 μM DNA

[NaCl] in mM	α 1 (%)	α 2 (%)	α 3 (%)	τ 1 (ps)	τ 2 (ns)	τ 3 (ns)	〈τf〉 (ns)	χ ^2
a Standard deviation for the fitting analysis is = ±5%.
0	94.47	1.23	4.30	331	3.87	15.28	10.15	1.04
100	92.42	3.50	4.08	334	4.63	14.45	9.41	1.11
200	90.61	5.68	3.71	339	5.87	13.56	8.22	1.09
300	88.34	8.02	3.64	345	6.34	12.89	7.43	1.13
500	85.57	10.97	3.46	351	7.11	12.13	7.20	1.12

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To obtain additional insight into the microenvironment around the drug, a time-resolved fluorescence anisotropy decay study of 9-MA in aqueous buffer and in the DNA environment has been performed. The anisotropy decay profiles of the drug within the microenvironment of DNA, both in the absence and the presence of NaCl are presented in Fig. 5a. The fluorophore demonstrates straightforward single exponential anisotropy decay with a reorientation time of ∼100 ps in aqueous buffer medium.¹⁹ However, in DNA medium, the drug demonstrates an interesting as well as not-so-common “dip-and-rise” decay pattern (due to the presence of a growth component at intermediate time) with the prominence of the aforesaid pattern being enriched with increasing ionic strength of the medium. Such a dip-and-rise kind of profile is treated as a characteristic for the concurrence of at least two populations having significantly different values of both fluorescence lifetime and rotational correlation time.^24,27–29

Fig. 5 (a) Fluorescence depolarization profile of the drug 9-MA in various environments as indicated in the figure legend. (b) Circular dichroic spectral profile of DNA (50 μM) with increasing 9-MA concentration. Curves i → v correspond to [9-MA] = 0, 2.8, 5.7 and 10.8 μM.

This kind of time-resolved anisotropy behavior can be explained with the help of an associated exponential model proposed by Ludescher et al.,²⁸ in which the fluorescence lifetime and amplitude of the total intensity decay components are linked specifically with individual anisotropy parameters as follows:

where,in which f_i(t) is the time-dependent weighing factor, I_T(t) is the total emission intensity decay, θ_i is the ith rotational correlation time, α_i, τ_i are the ith fluorescence amplitude and lifetime, respectively, and r(0) is the initial pre-rotational anisotropy. Now, it is obvious that the aforementioned dip-and-rise anisotropy profile condition is best realized for systems having a reasonably low lifetime under unbound conditions (i.e. in aqueous buffer), where internal motions favor rapid non-radiative deactivation of the emissive state, whereas under DNA-bound conditions when the internal motions are severely restricted, a noteworthy enhancement of lifetime takes place which is the case here. We have thus attributed the faster motion to the solvent exposed (free) drug and the slower motion to the bound counterpart (rotation of the drug along with a part or the whole of the macromolecule), with the obvious fact that the population of the DNA-bound form of the drug will be progressively encouraged with increasing NaCl concentration (as also has been observed in the time resolved emission decay measurements), resulting in the prominent appearance of the dip-and-rise pattern in the anisotropy decay profiles.

Conformation investigation: circular dichroism spectroscopy. The perturbation in the secondary structure of DNA upon interaction with the drug molecule has been exploited by circular dichroism. The far-UV CD spectra (220–320 nm) of DNA in aqueous buffer exhibits a typical shape (videFig. 5b), revealing a minimum at ∼247 nm and a maximum at ∼276 nm corresponding to the right handed B-form.^20,30 The influence of the interaction of DNA with 9-MA on the secondary structure of the biomolecule has been followed by monitoring the far-UV CD spectra of DNA in the presence of increasing concentration of the drug and the relevant spectra are displayed in Fig. 5b, where an enhancement in the CD signal of DNA with increasing concentration of 9-MA is noteworthy. Via a direct comparison with the literature, the increase in the band intensity at ∼276 nm has been rationalized on the basis of disruption of the stacking contacts of the bases due to intercalation of the drug, which is required to accommodate the intercalated drug within a particular base pair.^19,29 This observation categorically establishes intercalation of the drug within the DNA helix since electrostatic or groove binding are known to hardly influence the CD signal of DNA.

Kinetic study of DNA–drug association and dissociation processes. In the study of the interaction of small drug molecules with relevant biological and biomimicking targets, both the kinetics of association and dissociation are considered to have crucial diagnostic significance. The kinetics of the 9-MA–DNA association reaction is studied by monitoring the enhancement of the drug fluorescence at λ_em = 470 nm upon interaction with DNA. Fig. 6a displays the representative fluorescence trace for the time course describing the aforesaid process. The fluorescence trace has been fitted by an exponential rise function according to a pseudo-first order kinetics model within the presently employed experimental window (the concentration of DNA in the solution under investigation was significantly larger than that of the drug). The corresponding nonlinear regression equation is as follows:

I(t) = A[1 − exp(−k_at)] + C (7)

in which I(t) is the fluorescence intensity at time t, A is the amplitude corresponding to the apparent association rate constant k_a and C is the fluorescence intensity at equilibrium. The drug–DNA association kinetics is found to be characterized by a rate constant k_a = (4.31 ± 0.08) × 10⁻² s⁻¹ at 298 K. On the other hand, surfactant-induced (we have used the cationic surfactant CTAB to ensure a better interaction with anionic DNA, the concentration used being 0.2 mM which is sufficiently low for a significant interaction between the drug and the surfactant) dissociation kinetics of the drug is monitored by the fluorescence decrement of the DNA-bound drug at the same wavelength (videFig. 6b) using a simple mono-exponential decay function (here also we have used the pseudo first order reaction model), which was found to be much slower, characterized by a dissociation constant k_d = (2.13 ± 0.09) × 10⁻³ s⁻¹. However, understanding the fact that the kinetic rate constant of dissociation determined in the presence of the surfactant cannot be directly compared with the dissociation rate constant in the absence of the same, we have used the following expression to estimate a value of the dissociation rate constant:in which the equilibrium constant is taken to be K_a = (5.84 ± 0.4) × 10⁴ M⁻¹ (videFig. 2a), and k₁ and k₋₁ indicate the association and dissociation rate constants, respectively. The dissociation rate constant thus determined comes out to be k₋₁ = (1.28 ± 0.8) × 10⁻⁶ M⁻¹ s⁻¹. Thus, the data imply that with the studied drug molecule, 9-MA, the prerequisite of relatively faster association kinetics with DNA and slower dissociation kinetics from DNA can be achieved which demonstrates the aforesaid molecule to be able to function as an efficient therapeutic agent.^16,25 However, to obtain further details on the mechanism of the association/dissociation kinetics, more explicit experiments, including dependence of the rate constants on relevant extrinsic/intrinsic factors are required. In the present context, we refrain from making more elaborate comments on the issue, given the aim of the present work.

Fig. 6 Fluorescence kinetic profile for the (a) drug (9-MA)–DNA binding interaction and (b) surfactant (CTAB) induced dissociation of the drug from DNA. The represented profile describes the time course of fluorescence enhancement of 9-MA upon interaction with DNA and fluorescence decrement of the DNA-bound drug in the presence of the surfactant (CTAB) respectively. The grey lines designate the raw data and the blue lines are the fitted lines ([9-MA] = 2 μM, [DNA] = 100 μM and [CTAB] = 0.2 mM).

Conclusion

The present study reports a spectral exploration of the binding interaction of a potent chloride channel blocker, 9-MA, with calf-thymus DNA. The photophysical and dynamical behavior of the drug molecule are found to be remarkably modified as a result of interaction with DNA in comparison with those in the aqueous buffer phase. From the sequence of studies undertaken in the present contribution, the mode of binding of 9-MA with DNA has been evinced to be principally intercalative, coupled with some contribution from groove binding, the extent of which enhances rapidly with the increase in ionic strength of the medium. Further, with an eye to assess the efficacy of the drug to function as a therapeutic agent, the influence of drug binding on the secondary structural contents has been evaluated from CD measurements. The relatively slow rate of dissociation of the drug from the target (as compared to the association process) also demonstrates the therapeutic worth of the molecule.

Overall, with a view to the promising prospects of chloride channel blocker drugs on the biological field, the characterization of the strength, mode, and dynamics of binding of the drug 9-MA with DNA is of potential importance and hence, forms a demanding avenue of research. Therefore, it is perhaps not exaggerating to state that the results of the present study are of potential to yield a qualitative insight into the pharmacokinetic behavior of the drug which could be further extended to other bio-molecules from the viewpoint of understanding the biological applications of the drug.

Acknowledgements

AG and SG gratefully acknowledge Senior Research Fellowships respectively from CSIR and UGC, New Delhi, Govt. of India. NG likes to acknowledge UPE and CRNN, CU and DST, India, for financial assistance. We acknowledge the instrumental facilities of Indian Association for the Cultivation of Science, India, for providing access to CD measurements.

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