Research article Received: 07 October 2015,
Revised: 07 January 2016,
Accepted: 15 February 2016
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/bio.3126
Spectroscopic, electrochemical and molecular docking studies of dothiepin and doxepin with bovine serum albumin and DNA base N Rajendiran* and J Thulasidhasan ABSTRACT: The interaction of dothiepin (DOT) and doxepin (DOX) with bovine serum albumin (BSA) and a DNA base (adenine) was studied using UV–visible, fluorescence, attenuated total reflection–infra-red (ATR-IR), cyclic voltammetry and molecular docking methods. Strong fluorescence quenching was observed upon interaction of DOT and DOX with BSA/adenine and the mechanism suggested static quenching. Hydrophobic and hydrogen bonding interactions were the predominant intermolecular forces needed to stabilize the copolymer. Upon addition of the drugs: (i) the tautomeric equilibrium structure of the adenine was changed; and (ii) the oxidation and the reduction peaks of the adenine/BSA interaction shifted towards high and low potentials, respectively. In ATR-IR, the band shift of amides I and II indicated a change in secondary structure of BSA upon binding to DOT and DOX drugs. The reduction in voltammetric current in the presence of BSA/adenine was attributed to slow diffusion of BSA/adenine binding with DOX/DOT. The docking method indicated that the drug moiety interacted with the BSA molecule. Copyright © 2016 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site. Keywords: dothiepin; doxepin; BSA; quenching; cyclic voltammetry; molecular docking
Introduction A huge increase in research to characterize various potential drugs interactions with different biological and bio-imitator assemblies has recently been observed. This has resulted in the confident prediction of related planned assemblies via biological, photochemical, and photophysical processes (1). Bovine serum albumin (BSA), a major soluble protein, serves as a transporter for a variety of endogenous and exogenous ligands such as fatty acids, steroids, drugs, metal ions and metabolites (1). Over the last few years, BSA has been used widely as a model protein because of its structural homology with human serum albumin (HSA) (2). BSA has 80% structural similarity to HSA with only a major difference in the number of tryptophans: HSA has only one tryptophan, whereas BSA has two. BSA has been the usual selection for protein binding studies because of its abundance, low cost, ease of purification, stability, medical importance and drug-binding properties (3–5). Many drugs, particularly those used as local anaesthetics, tranquillizers, antidepressants, and antibiotics, exercise their action by interactions with biological membranes. These compounds must be carried to their action site and, usually, this is achieved by globular protein serum albumins (blood carrier proteins) to which they bind with different affinities. Tricyclic antidepressant drugs (TCAs) are one of the largest groups of drugs used for the treatment of psychiatric disorders such as depression, mainly endogenous major depression. The function of these drugs is to block the re-uptake of neurotransmitters and serotonin in the central nervous system (6). The chemical structures of selected TCA compounds [dothiepin – (11(16H)-(3[dimethylamino]propylidene)dibenz[b,e]thiopine and doxepin – (11(16H)-(3-[dimethylamino]propylidene)dibenz[b,e]oxepine] are shown in Fig. 1. The importance of TCAs is highlighted not only
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by their therapeutic use for depressive disorders but also by their use in a variety of diseases that have an effect on mental state – such as insomnia, anxiety disorder, post-traumatic stress disorder, obsessive compulsive disorder, and chronic pain (7,8). Furthermore, there is an increasing field related to cognitive enhancing and life style use of TCA (9). The analysis of these compounds, therefore, is important for quality assurance in pharmaceutical preparations and to obtain optimum therapeutic concentrations to minimize the risk of toxicity. Various studies on serum albumins that involve the binding of small molecules, in particular fatty acids and drugs, based on different techniques (UV–visible, fluorescence spectroscopy, ATR-IR, Raman spectroscopy, electrochemistry, NMR, etc.) have been described previously (10–13). When these molecules bind to a serum albumin, intramolecular forces that are mainly used to sustain secondary structure can be altered, developing conformational changes in the protein (14). Drug interactions at the protein binding level notably affect important factors such as drug availability, efficacy, transport, elimination rate, etc. Hence, studies on this aspect can furnish information on the structural features that influence the
* Correspondence to: N. Rajendiran, Department of Chemistry, Annamalai University, Annamalai Nagar 608 002, Tamilnadu India. E-mail:
[email protected] Department of Chemistry, Annamalai University, Annamalai Nagar, 608 002, Tamilnadu India Abbreviations: ATR, attenuated total reflection; BSA, bovine serum albumin; HSA, human serum albumin; IR, infra-red; LGA, Lamarckian genetic algorithm; LW, longer wavelength; SD, standard deviation; SW, shorter wavelength
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N. Rajendiran and J. Thulasidhasan
Figure 1. Chemical structures of (a) doxepin and (b) dothiepin.
therapeutic effectiveness of drugs and have been an interesting research field of study in life sciences, chemistry and clinical medicine (15). In particular, our investigation is focused on: (i) comparison of the quenching of BSA and adenine with two TCAs drugs; (ii) determination of binding constant and number of binding sites of the systems; (iii) determination of FRET – energy transfer and binding distance between proteins and drugs; (iv) analysis of the oxidation and reduction potential of the drugprotein molecules; and (v) molecular docking analysis and conformation changes of BSA and adenine upon binding to drugs.
Spartan ‘08 software and then optimised by the semi-empirical PM3 method. The MMFF94 force field was used for energy minimization of the drug (DOX and DOT) molecules using the docking server (17). Gasteiger partial charges were added to the drug atoms. Non-polar hydrogen atoms were merged, and rotatable bonds were defined. Docking calculations were carried out on a 4F5S protein model. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the aid of Auto Dock tools (18). Affinity ( grid) maps of 20 × 20 × 20 Å grid points and 0.375 Å spacing were generated using the Auto grid program (18). Auto Dock parameter set- and distance-dependent dielectric functions were used in the calculation of the van der Waals forces and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method. Initial position, orientation, and torsions of the drug molecules were set randomly. Each docking experiment was derived from 10 different runs that were set to terminate after a maximum of 25 × 104 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of five were applied.
Experimental Results and discussion
Materials Doxepin (DOX), dothiepin (DOT), adenine and BSA were obtained from Sigma-Aldrich chemical company (USA) and used without further purification. All other reagents were of analytical grade. The purity of the compound was checked by similar fluorescence spectra when excited with different wavelengths. Triple-distilled water was used for the preparation of aqueous solutions. BSA solutions were prepared in 2 × 103 M Tris–HCl buffered at pH 7.4. The concentration of the drug solutions was varied from 1 × 103 to 10 × 102 M; 0.2 mL of drug in methanol solution was used for all binding experiments. All solutions were stored in a refrigerator at 4 °C in the dark. Instruments Absorption spectral measurements were carried out with a UV– visible spectrophotometer (model UV-2600 Shimadzu, Japan) and fluorescence measurements were performed on a spectrofluorophotometer (model RF-5301PC, Shimadzu, Japan) equipped with 1.0 cm quartz cells. The excitation wavelength was fixed for BSA with drug at 280 nm and for adenine with drug at 270 nm. Cyclic voltammetry measurements were performed through an electrochemical workstation (model CHI 620D, CH Instruments, USA) with a three electrode system: surface area 0.1963 cm2 platinum disc as working electrode; saturated silver electrode as reference electrode; and a platinum foil as counter electrode. Prior to use, the working electrode was polished with 0.05 μm alumina and thoroughly washed in an ultrasonic bath for 5 min. Before experiments, the solution within a singlecompartment cell was deaerated by purging with pure N2 gas for 5 min. The pH values in the range 2.5–11.5 were measured on an Elico pH meter model LI-120. Molecular docking Molecular docking calculations were carried out using an online docking server (http://www.dockingserver.com) (16). The initial geometries of DOX and DOT were constructed with the aid of
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Absorption measurements UV–visible absorption spectroscopy was employed to investigate the structural changes of the proteins and to work out the complex formation between the drugs and proteins. Absorption spectra of BSA and adenine with and without DOX and DOT drug concentrations are given in Table S1 and Fig. S1. Without BSA and adenine, the absorption maxima of DOT was seen at 300, 260, 218 nm, and for DOX was seen at 292, 245s, and 210 nm, respectively (19,20). The absorption maximum of adenine appeared at 260 nm and for BSA was observed at 280 nm (Fig. S1) and is mainly due to the presence of the tryptophan and tyrosine residues in BSA. With increasing concentrations of TCA drugs, the absorbance of BSA and adenine decreased in a regular manner (Fig. S1). There was no marked UV–visible spectral shift observed in BSA and adenine when varying the drug concentration, except a decrease in the absorption maxima. The changes in the absorption spectra indicated that there was interaction of the TCA drugs with BSA and adenine to form protein–drug complexes (21–23). Fluorescence measurements The emission spectra of BSA and adenine in TCA drugs were more pronounced than the absorption spectra. Figure 2 and Table S1 show the fluorescence spectra of BSA and adenine with different concentrations of the TCA drug molecules. In the absence of BSA/adenine, dual fluorescence was observed in the TCA drug molecules (DOT ~336, 384 nm, and DOX ~324, 362 nm). When the concentration of DOX was increased, the fluorescence intensity of BSA (at ~338 nm) decreased with increasing longer wavelength (~458 nm; Fig 2). An isoemissive point at ~415 nm indicated that these spectra were linear combinations of two components and reflected equilibrium between these components, providing evidence for the interaction of BSA with DOX (24). At the same time, the emission intensity of BSA was decreased at ~338 nm without longer wavelengths by increasing the DOT concentrations.
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Studies of dothiepin and doxepin with BSA and DNA base
5
Figure 2. Fluorescence spectra of BSA and adenine with different DOX and DOT concentrations (× 10 280 nm for BSA and 270 nm for adenine. Insets: fluorescence intensity versus drug concentration.
The fluorescence spectra of aqueous adenine solutions as a function of various concentrations of TCA drugs are presented in Fig. 2. For adenine alone, three emission maxima appeared at 430, 330 and 288 nm, whereas a single emission was observed (338 nm) in the BSA molecule. When drug concentration was increased, there was no significant spectral change observed in BSA (338 nm), but red or blue shifted emission spectra were noticed in adenine (330–340 nm). Interestingly, when the DOX and DOT concentrations were increased, the shorter wavelength emission intensity decreased while the longer wavelength emission intensity increased. The fluorescence bathochromic and hypsochromic spectral shifts indicated that both TCA drug molecules interacted with BSA and adenine. The changes in the fluorescence spectrum indicated the formation of a protein–drug complex. It is well known that adenine forms a number of tautomer compounds (Fig. S2) that can be quickly inter-changed and are usually considered equivalent [20]. The normal Stokes shifted emission (F1) is thought to originate from the excitation of the normal adenine molecule. The F2 and F3 emissions are taken to originate from tautomer I and tautomer II. Therefore the F1 emission maxima for adenine are the same with both drug molecules, whereas the F2 and F3 emission maxima are different, as adenine binds to the drugs via hydrogen bonds to assist in stabilizing the nucleic acid structures. The steady decrease and increase in the adenine emission yield with the addition of drugs may have two causes: (i) ground state trapping of transferable proton within the adenine molecule; the N-H proton may not be accessible to the nitrogen atom of the heterocyclic; and (ii) non-radiative decay of the tautomer; interaction of drug with amine or the –NH– or the –N = group of the adenine molecule. This non-radiative channel is expected to decrease or diminish the fluorescence yield of the tautomer form.
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M): (1) 0, (2) 1, (3) 3, (4) 5, (5) 7, (6) 9, and (7) 10; excitation wavelength:
We also compared the interaction of the above drugs with cyclodextrin (CD), described below. The absorption and fluorescence attributes of TCA drugs have been seen to undergo extreme changes in the vicinity of CD (19,20). Upon addition of CD, in the TCA drug molecules, the emission band at ~340 nm increased in a regular manner with appearance of longer wavelength emission at 430 and 455 nm. In the presence of CD, two major changes were observed in the S1 state: (i) fluorescence emission intensity increased with CD concentrations; and (ii) the emission spectrum was observed along with a large red shift. In both drug molecules, hydrophobic interactions and van der Waals forces are presented in the CD cavity, and a controlled spectrum was observed because the sizes of the two drugs are larger than CD hollow space. Furthermore, at higher CD concentrations, viscosity variations may play a vital role in producing controlled spectrum in the CD solutions. The CD-dependent emission spectra showed that the longer wavelength (LW) band is more responsive to CD concentration, while the shorter wavelength (SW) band showed a small enhancement with a standard bathochromic shift. In water, LW intensity was very low. With the addition of CD, both LW and SW intensities were raised. However, the rate of enhancement of the LW emission was larger than that for the SW band. Inside the CD cavity, drug molecules encounter a less polar environment and the geometrical restrictions of the CD hollow space would restrict the free rotation of the aliphatic chain. However, on addition of the drugs into the BSA and adenine molecules, the emission maxima of the drug molecules were completely lost. The above results showed that the hydrophobic, electrostatic and hydrogen bonding interactions play a major role in Tricyclic antidepressant drugs (TCA) interactions drugs with BSA and adenine.
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N. Rajendiran and J. Thulasidhasan Table 1. Stern–Volmer quenching constant (Ksv), modified Stern–Volmer association constant (Ka) and bimolecular quenching rate constant (Kq) of adenine and BSA with DOX and DOT at 300 K; λex = 280 nm for BSA, 270 nm for adenine; λem = 290–500 nm, (pH ~7.4) Drugs S–V quenching
Adenine BSA
Modified S–V quenching
Adenine BSA
Ksv (M1) 5
Kq (M1 sec1) 13
Ra
SD
DOX DOT DOX DOT
1.22 × 10 7.42 × 104 1.35 × 105 1.27 × 105
1.22 × 10 7.42 × 1012 1.35 × 1013 1.27 × 1013
0.990 0.991 0.999 0.999
0.492 0.668 0.592 0.761
Drugs DOX DOT DOX DOT
Ka (M1) 9.71 × 104 7.28 × 104 1.37 × 105 1.02 × 105
ΔG0 (kJ/mol) 19.15 19.02 20.93 20.78
n 0.789 0.921 0.988 0.954
Ra 0.996 0.999 0.999 0.998
n, number of binding sites; Ra, linear correlation coefficient; SD, standard deviation.
Quenching mechanism The quenching effect of the drugs with BSA is higher than that of adenine (Table 1), which was further analysed by Stern– Volmer equations. The possible quenching mechanism can be interpreted by the fluorescence-quenching spectra of BSA and adenine. In these cases, with increasing the TCA drug concentrations, the fluorescence intensity decreased in a regular manner. This result suggests that both drug molecules can bind to BSA and adenine. The fluorescence-quenching data were analysed using the Stern–Volmer equation (see Supporting information, eqn S1). Figures 3 and S3 display the Stern–Volmer and modified Stern-Volmer plots of BSA/adenine with various concentrations of TCA drugs. As can be seen in Fig. 3 and Fig. S3, the Stern–Volmer plots are linear, which indicates that the quenching process is static. In general, the fluorescence lifetime (τ0) of BSA is approximately 108 sec (25) and the Ksv value is obtained from the slope of the linear plot. The quenching constants (Kq) of the drugs was determined (Table 1) using eqn 1. The obtained Kq values are in the range of 1012 L mol1 sec1, which far exceed the diffusion controlled rate constant in aqueous solution, i.e. 2 × 1010 L mol1 sec1 (26), confirming that quenching does not engage the dynamic diffusion method but happens statically in the complex. Both plots showed a relatively good linear relationship, which indicated that the dominating quenching system is not dynamic but static. The binding constant (Ka) and binding sites (n) can be obtained using fluorescence intensity data. Drug molecules bind separately to a set of equivalent sites on a protein molecule and the equilibrium between free and bound molecules (interaction complex) is given (27) by eqn (S2). Ka and n values were obtained from the intercept and slope of the plot of log[(F0 - F)/F] versus log[Q] (Figs 3c and S3c). The n values for the BSA–TCA system were 0.79 and 0.92 for DOX and DOT, respectively ( Table 1). The binding sites values are very close to unity, which indicated that only one independent class of binding site is present in BSA and adenine with TCA drugs. The Ksv value for BSA/adenine with DOX is higher than that for the DOT drug molecule. Furthermore, the Ksv value of BSA–DOX is comparatively more than that for adenine–DOX, and suggests that BSA is more quenched than adenine.
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Figure 3. (a) Stern–Volmer and (b) modified Stern–Volmer plots for quenching of BSA fluorescence by DOT and DOX. (c) Plots of log [(F0 F)/F] versus log [Q] for 5 DOT and DOX quenching effect on BSA fluorescence at 300 K. CBSA = 2.0 × 10 M; pH 7.4; λex = 280 nm, λem = 290–500 nm.
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Studies of dothiepin and doxepin with BSA and DNA base Nature of the binding forces and free energy change Differences in Gibbs free energy (ΔG) can be derived using the following eqn 1 and binding constant (Ka): ΔG ¼ lnK a RT
(1)
where, Ka is the binding constant at a corresponding temperature, R is the gas constant and T is the absolute temperature; the values are given in Table 1. The negative ΔG value indicated the spontaneity of the binding between the drugs and BSA. The ΔG value for the BSA–drug was more negative than that for adenine–drug, which indicated that the interaction of BSA with the drugs was more spontaneous than that for adenine and drugs. The above results and the fluorescence-quenching results indicated that both TCA drugs interacted with BSA/adenine. Binding distance from fluorescence resonance energy transfer (FRET) According to FRET (28), the rate of energy transfer depends on: (i) the distance between the donor and the acceptor; (ii) the relative orientation of the donor and acceptor dipoles; and (iii) the extent of overlap of the emission spectrum of the donor with the absorption spectrum of the acceptor. The energy transfer effect is related not only to the distance between the acceptor and the donor, but also to the critical energy transfer distance R0. The dipole orientation factor (k2) is the least definite factor in calculation of the critical transfer distance, R0. If the donor and acceptor fall rapidly and are free to assume any orientation, then k2 equals 2/3 (29). On the basis of eqns (S3–S5), the following parameters were obtained for BSA–DOX and BSA–DOT, respectively: J = 1.66 and 1.55 × 1014 cm3 L mol1, R0 = 0.71 and 0.66 nm, E = 0.35 and 0.34, r = 1.57 and 0.73 nm (Table S1). According to conditions of Foster’s non-radioactive energy transfer theory, a static quenching interaction between BSA and TCA drugs could be confirmed. Table S1 shows the energy transfer efficiency (E) for the TAC drugs with BSA/adenine; the fact that DOX binds more efficiency to the active site of BSA than do other TCA drugs further supports the above implication. Attenuated total reflection–infra-red spectroscopy Attenuated total reflection (ATR) is a sampling technique used in conjunction with infrared (IR) spectroscopy that enables samples to be examined directly in the solid or liquid state without further preparation (30–34). An IR spectrophotometer is mainly used to estimate the structure of organic compounds. In order to obtain detailed information on the BSA conformation after interaction between BSA and the drugs, we recorded the ATR-IR spectra of DOX, DOT, free BSA and its interaction with the drugs and are shown in Fig. 4. The peak near 1650 cm1 in Fig. 4(a) is the amide I band. This result is based on assumptions from the C = O stretching vibrations of the peptide bond (35). The peaks near 1540 cm1 (N–H bending vibration/C–N stretching vibration) and 1275 cm1 (C–N stretching vibration/N–H bending vibration) are assumed to be amide II band and amide III band, respectively. The peak near 3300 cm1 is assumed to be the N–H bending vibration and the peak near 1400 cm1 results from the protein COO side chain. As the absorption
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Figure 4. IR spectra of (a) BSA, (b) DOX, (c) DOT, (d) BSA-DOX and (e) BSA-DOT.
peak position and the shape of the amide I band differ according to the secondary structure, peak analysis can yield information on the secondary structure. The aromatic CH stretching frequency for DOX and DOT appeared at 3424 and 3432 cm1 respectively and is moved to a lower frequency with BSA. The N–CH3 stretching frequencies of DOX and DOT appeared at around 2955, 2951 cm1 and was moved to ~2942 and 2978 cm1 respectively. Furthermore, the asymmetry frequency of CH3 seen at 1479 and 1468 cm1 were altered as a result of their interaction with BSA. The aromatic C = C bending vibrations at ~1633, and 1632 cm1 moved to longer stretching frequencies in BSA–drug complexes. In addition, the ratio between the pure drug molecule stretching frequency intensities varied mostly varied drug:BSA. Furthermore, the amide I bands of the BSA-drug system shifted for DOX from 1649 to 1655 cm1 and for DOT moved from 1649 to 1658 cm1 respectively. Similarly the amide II bands shifted from 1545 to 1562 cm1 and 1532 cm1 for DOX and DOT respectively. The above results indicated that there was a change in secondary structure of BSA upon binding to the drugs. Cyclic voltammetry studies Figure S4 and Table 2 show the cyclic voltammograms for BSA and adenine in the absence and presence of different concentrations of DOX and DOT. Curve 1 represents the oxidation curve of DOX (Epa = 1.17 V) and redox peak curve of DOT (Epa = 1.096 and Epc = 0.520) drugs respectively (at Glassy carbon electrode
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N. Rajendiran and J. Thulasidhasan Table 2. CV for adenine/BSA with DOX and DOT (scan rate, 100 mV sec1, concentration of adenine and BSA 2 × 106 M; drugs concentrations: 0, 3, 7 and 10 × 105 M) Drug–BSA or adenine DOX only DOT only BSA only Adenine only BSA–DOX
BSA–DOT
Adenine–DOX
Adenine–DOT
Drug concentration 1 × 103 1 × 103 2 × 105 2 × 105 3 × 104 7 × 104 10 × 104 3 × 104 7 × 104 10 × 104 3 × 104 7 × 104 10 × 104 3 × 104 7 × 104 10 × 104
Epa
Ipa
1172 1096 973 987 1234 941 921 1010 0.706 990 701 980 1544 841 1318 1423
1.328 1.277 0.524 0.613 0.940 0.766 0.713 0.873 – 0.493 0.597 0.880 1.568 0.802 1.866 2.094
Epc
Ipc
– 520
– -1.960
1190 1220 1225 – – – 1197 1178 1040 – – –
0.706 0.760 0.841 – 431 – 1.091 1.212 1.293 – – –
Epa - Epc/2
Ipa/Ipc
586 808 487 678 1212 1080 1073 505 – 495 939 1079 1292 420 659 711
– 1.534 – –
– – 1.827 1.377 0.824 – – –
1
Figure 5. Cyclic voltammograms of BSA in presence of (a) DOX and (b) DOT at different scan rate (100 to 600 mV s ) and adenine in presence of (c) DOX and (d) DOT at different 1 3 scan rate (100 to 600 mV s ), Concentration of drugs = 1 x 10 M.
(GCE)). In curve 2, it can be seen that in the absence and presence of drugs, BSA displayed oxidation waves at the glassy carbon electrode (pH ~7.4). On addition of BSA, the oxidation peak current (Ipa) of the
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drugs (DOX and DOT) largely decreased with positive and negative peak potential. The oxidation peak shifted towards lower potentials and an increasing oxidation current was observed. When more
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Studies of dothiepin and doxepin with BSA and DNA base drugs added to BSA, the peak current (Ipa) decreased. In the adenine–drug system, the oxidation peak current (Ipa) increased and the reduction peak current (Ipc) of DOX with adenine decreased. With the addition of BSA to these drugs, the voltammetric oxidation peak currents decreased or increased and positively shifted, which indicated that there were interactions between the drugs and BSA/adenine. The drop of the voltammetric reduction current in the presence of BSA/adenine may be attributed to slow diffusion of the BSA/adenine binding with TCA drugs. The results also specified that interactions between drugs and BSA/adenine happened in the reaction and that the electrode process was irreversible (21–23). Cyclic voltammograms of TCA drugs with BSA and adenine solution (pH ~7.4) with the effect of scan rate on the peak current were investigated. The cyclic voltammograms with scan rate (Fig. 5) show that the electrode reactions of the drugs with BSA and adenine are an irreversible processes. The peak current was proportional to the root of scan rate over a range of 100 to 600 mV sec1. The reductive reaction of the drugs–BSA/adenine solution had the characteristics of strong adsorption and emission behaviour and the irreversible electrode process. Laviron’s equation may be
used to evaluate the kinetic constants of the electrode reaction in the absence and presence of protein: Ep ¼ E0 þ RT=ðαnFÞ ½ lnðRTksÞ=ðαnFÞ – lnν
(2)
where α is the electron transfer coefficient, ks the standard rate constant of the surface reaction, υ the scan rate, E0 the formal potential and n the electron transfer number. Parameters of these drug reaction with BSA/adenine were calculated using eqn (2), and the results are shown in Figs S5 and S6. The plot of Ep versus υ is a well defined straight line and the ‘αn’ value can be calculated from the slope and ks from the intercept. The plot of Ep versus ln υ was non-linear (Figs S7 and S8). From the slope, αn values of TCA drugs can be determined, and from the intercepts, the ks values can be calculated. E0 values of the TCA drugs can be determined from Figs S5 and S6 on the ordinate by extrapolating the line to υ = 0. Measurement of stoichiometry of BSA- drug complex According to Li and Min, (36) the composition and the equilibrium constant can be calculated based on changes in peak current (Ip). It
6
Figure 6. Linear plot of log[drug] versus log[ΔI/(ΔImax - ΔI)]. (a) DOXBSA, (b) DOTBSA, (c) DOXadenine, and (d) DOTadenine. (BSA and adenine concentration, 2 × 10 ).
Table 3. Estimated free energy, inhibition constant, electrostatic energy and total intermolecular energy of BSA with DOX and DOT Drug
DOX DOT
Estimated free energy of binding (kcal/mol) 6.24 4.46
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Estimated inhibition constant (μM) 26.55 542.12
vdW + H-bond + desolv energy (kcal/mol) 7.01 5.39
Electrostatic energy (kcal/mol) +0.06 +0.08
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Total intermolecular energy (kcal/mol) 6.95 5.31
Frequency
30% 10%
Interact surface 730.76 808.17
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N. Rajendiran and J. Thulasidhasan was proposed that a single complex of drug–BSA/adenine was formed. The relationship of log[ΔI/(ΔImax - ΔI)] with log[Q] was calculated and is plotted in Fig. 6. From the intercept and slope, m and βs were deduced. This indicated that both drugs bound to BSA/adenine, i.e. formed BSA:drug and adenine:drug complexes.
Furthermore the plot of log[Q] versus log[ΔI/(ΔImax - ΔI)] was linear and indicated that BSA/adenine bound to TCA drugs. Furthermore, the plot of log[Q] versus log[ΔI/(ΔImax - ΔI)] was linear (Fig. 6), which indicated that these drugs formed BSA:drug and adenine: drug complexes with the proteins.
Figure 7. Best binding mode between (a) DOX and (b) DOT with BSA. The important residues of BSA are represented using lines and the ligand structure is represented using the ‘ball and stick’ format. Hydrogen-bonding plots between (c) DOX and (d) DOT with BSA. BSA residues are represented using black dots and the hydrogen-bonding interactions are represented using red dots. Colour of the atoms: skeleton structure – BSA; blue – nitrogen; red – oxygen; yellow – sulphur; green – carbon.
Figure 8. Two-dimensional schematic representation of hydrophobic interactions of BSA with DOX and DOT. Active residues are represents in green colour sweeps. Colour of the atoms: blue – nitrogen, red – oxygen, yellow – sulphur, black – carbon.
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Studies of dothiepin and doxepin with BSA and DNA base Molecular docking
References
Evaluation of the exact conformation of the drugs with BSA and identification of the precise binding site on BSA is very essential and important for understanding the proper functioning of the TCA drugs. The results shown in Table 3 and Fig. 7(a, b) revealed the most possible binding sites and positions for the drug molecules in BSA protein. The binding site of BSA was studied to understand the nature of the residues defining the site. Figure 7(c, d) shows the hydrogen bonding and hydrophobic interaction plots for BSA and TCA drugs and its corresponding two-dimensional schematic diagrams are shown in Fig. 8. The DOX molecule is surrounded by residues TYR400, ASN404, LYS524, GLN525, ALA527, LEU528, VAL546, MET547, PHE550, VAL551 and DOT molecule by LEU397, TYR400, ASN404, LYS524, MET547, GLN525, PHE550, VAL551, respectively. The drug core contact with the protein was anchored in the binding site by H-bonds. However, a series of hydrophobic residues, MET 547 (4.6054), TYR 400 (0.4877), ALA 527 (0.3886), LEU 528 (0.042), VAL 546 (0.0367), PHE550 (0.5807), VAL551 (0.7715), around the peripheral region of the molecule interacted with the DOX drug molecule through hydrophobic interactions. Furthermore, the following residues were more likely residues for the hydrophobic interaction in BSA–DOT system, TYR400 (0.4951), MET547 (0.5479), LEU397 (0.2729), VAL551 (0.3376), PHE550 (0.7508), GLU548 (0.4362), LYS524 (0.4466) (Fig. 7). From the docking analysis (Tables S2 and S3), we could assume that the hydrogen bonding, hydrophobic interactions and the polar contacts collectively constituted the primary force for binding of molecule. Table 3 shows the estimation of free energy, van der Waals and hydrogen bonding interactions of BSA with the TCA drugs. The interaction of DOX with BSA is more negative than with other drugs. The above results suggested that DOX interacts more strongly with BSA than does the DOT molecule.
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Conclusion The interactions between BSA and adenine and two TCA drugs (DOX and DOT) were investigated using different techniques. BSA and adenine fluorescence can be quenched by these drugs, which implies that both drugs can bind to BSA and adenine molecules. TCA drugs bind to BSA and adenine with a stoichiometric ratio of 1:1 and the protein–drug complexes are stabilized mainly by hydrophobic and van der Waals interactions. Compared with adenine, BSA contributed a substantially higher binding efficiency for the drugs. The Ksv value of BSA/adenine with DOX is higher than that for DOT drugs and BSA-DOX and is comparatively more than adenine–DOX, suggesting that BSA is more quenched than adenine. With the addition of the TCA drugs: (i) the tautomeric equilibrium structure of adenine was changed; (ii) the IR spectra of pure BSA and pure dugs differed from that of the BSA:drug complex; and (iii) oxidation and reduction peaks of adenine/BSA shifted towards high and low potentials, respectively. Molecular docking studies showed that the DOX drug molecule had a higher binding affinity for BSA than did DOT.
Acknowledgements This work was supported by the Council of Scientific Industrial Research [no. 01(2549)/12/ EMR-II], New Delhi, India and the University Grants Commission [F. no. 41–351/2012 (SR)], New Delhi, India.
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