Accepted Manuscript Spectroscopic and molecular docking studies on the interaction of the drug olanzapine with calf thymus DNA Nahid Shahabadi, Somayeh Bagheri PII: DOI: Reference:

S1386-1425(14)01533-9 http://dx.doi.org/10.1016/j.saa.2014.10.036 SAA 12851

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

10 May 2014 22 July 2014 13 October 2014

Please cite this article as: N. Shahabadi, S. Bagheri, Spectroscopic and molecular docking studies on the interaction of the drug olanzapine with calf thymus DNA, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.10.036

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Spectroscopic and molecular docking studies on the interaction of the drug olanzapine with Calf thymus DNA

Nahid Shahabadi1*, Somayeh Bagheri1

1

Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran. *Corresponding author: Tel / Fax: +98-831-8360795 E-mail: [email protected]

1

Abstract The present study investigated the binding interaction between olanzapine and calf thymus DNA (ct-DNA) using emission, absorption, circular dichroism, viscosity measurements and molecular modeling. Thermodynamic parameters (∆H < 0 and ∆S < 0) indicated that hydrogen bond and van der Waals play main roles in the binding of the drug to ct-DNA. Spectrophotometric studies of the interaction of olanzapine with DNA have shown that it could bind to ct-DNA (Kb=2×103 M-1). The binding constant is comparable to standard groove binding drugs. Competitive fluorimetric studies with Hoechst 33258 have shown that olanzapine exhibits the ability to displace the DNA-bound Hoechst 33258 indicating that binds strongly in minor groove of DNA helix. Furthermore, the drug induces detectable changes in the CD spectrum of ct-DNA as well as changes in its viscosity. All of the experimental results prove that the groove binding must be predominant. The results obtained from experimental data were in good agreement with molecular modeling studies. Keywords: Olanzapine, DNA interaction, Hoechst 33258, Groove binding, Spectrum.

2

1. Introduction Olanzapine

(Fig.

1)

is

2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno

[2,

3-b][1,5]

benzodiazepine and its empirical formula is C17H20N4S. This drug is structurally and pharmacologically similar to the atypical antipsychotic clozapine, and is an antipsychotic medication that affects chemicals in the brain [1]. Olanzapine has been evaluated as an adjunctive medication for the prevention and treatment of chemotherapy-induced nausea and vomiting (CINV) in patients with cancer. Olanzapine’s activity at multiple receptors, particularly at the D2 and 5hydroxytryptamine-3 (5-HT3) receptors, which appear to be involved in nausea and emesis, suggests that it may have significant anti-emetic properties [2 , 3]. Understanding the binding of small molecules to DNA is potentially useful in developing principles to guide the synthesis of new improved drugs which can recognize a specific site or conformation of DNA [4, 5]. Generally, durg upon binding to DNA are stabilized through a series of weak interactions such as the п-stacking interactions of aromatic heterocyclic groups between the base pairs (intercalation), hydrogen bonding and van der Waals interactions of functional groups bound along the groove of the DNA helix [6,7]. Identifying which of the above interaction modes is operative for the molecule concerned, as well as which is predominant among the series of the weak interactions, is essential in designing an effective new drug and a DNA-probe molecule. In our laboratory, we have focused our attention to drugs and their metal complexes that target cellular DNA, to understand the mechanism of action at the molecular level, and also to investigate the effect of drugs as anticancer agents. In the present study, the interaction of olanzapine with calf thymus DNA was investigated in vitro using electronic absorption spectroscopy [8], fluorescence spectroscopy [9], circular dichroic spectral [10], viscosity measurements [11] and molecular modeling. Determination of the binding mode and affinity between the constituent molecules in molecular recognition is crucial to understanding the interaction mechanisms and to designing therapeutic interventions. 3

2. Experimental 2.1. Reagents. Olanzapine, ct-DNA and Tris–HCl were purchased from Sigma. Experiments were carried out in Tris–HCl buffer at pH=7.4. The buffer solution was prepared from Tris–(hydroxymethyl) aminom ethane, double distilled water and hydrogen chloride and pH was adjusted to 7.4. Stock solution of ct-DNA was prepared by dissolving about 2 mg of ct-DNA fibers in 2 mL Tris–HCl buffer (10 mM) by shaking gently and stored for 24 h at 4 °C and used within 5 days. The concentration of ctDNA solution was expressed in monomer units, as determined by spectrophotometry at 260 nm using an extinction coefficient (ߝp) of 6600 M-1C-1. A solution of ct-DNA in the buffer gave a ratio of UV absorbance at 260 and 280 nm of~1.8-1.9:1, indicating that the ct-DNA was sufficiently free from protein [12, 13]. Stock solution of Olanzapine was prepared by dissolving 0.0012 mg of the drug in 2.0 mL of Tris-HCl buffer (10 mM) (final concentration 10 -3 mol L-1). Aliquots of the ctDNA solution were treated with the drug at several input molar ratios (ri = [DNA]/ [drug]) at a constant drug concentration. The final volume of samples was made up with Tris–HCl (10 mM) and the samples were incubated at 25

for 1 h.

2.2. Instrumentation Absorbance spectra were recorded using an hp spectrophotometer (Agilent 8453) equipped with a thermostated bath (Huber polysat cc1). Absorption titration experiments were conducted by keeping the concentration of drug constant (5× 10-5) while varying the DNA concentration from 0 to 1× 10-4 M (ri = [DNA]/[drug] = 0.0, 0.3, 0.5, 0.8, 1.2, 1.7, 2). Absorbance values were recorded after each successive addition of DNA solution, followed by an equilibration period. Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200). The competitive interaction between the Hoechst 33258 and the olanzapine with DNA was carried out

4

as follows: fixed amounts of the Hoechst 33258 and DNA were titrated with increasing amounts of olanzapine solution. CD measurements were recorded on a JASCO (J-810) spectropolarimeter by keeping the concentration of DNA constant (5× 10-5) while varying the drug concentration (ri= [drug]/[DNA] = 0, 0.6, 0.8,0.9). Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) that was maintained at 25±5 ℃ using a constant temperature bath. The DNA concentration was fixed at 5×10-5 M, and flow time was measured with a digital stopwatch. The mean values of three replicated measurements were used to evaluate the viscosity of the samples. The values for relative specific viscosity(η/η0)1/3 where η0 and η are the specific viscosity contributions of DNA in the absence (η0) and in the presence of the drug (η), were plotted against ri (ri = [drug]/ [DNA]) [14]. 2.3. Molecular docking study MGL tools 1.5.4 with AutoGrid4 and AutoDock4 [15, 16] were used to set up and perform blind docking calculations between drug and DNA sequence. CT-DNA sequence d(CGCGAATTC GCG)2 dodecamer (PDB ID: 1BNA) obtained from the Protein Data Bank. Drug structure obtained from the Drug Bank (Drug ID: DB00334) was used for the docking studies. Receptor (DNA) and ligand (drug) files were provided using AutoDock Tools. First of all the heteroatoms including water molecules were deleted. The DNA was enclosed in a box with number of grid points in x×y×z directions, 94×104×126 and a grid spacing of 0.375 A˚. Lamarckian genetic algorithms, as implemented in AutoDock, were employed to perform docking calculations. All other parameters were default settings. For each of the docking cases, the lowest energy docked conformation, according to the AutoDock scoring function, was selected as the binding mode. The output from AutoDock was rendered with PyMol [17].

5

3. Results and discussion 3.1. UV–vis spectroscopy UV–vis absorption measurement is an effective method in detecting the binding strength and the mode of drug binding with ct-DNA. When a drug interacts with ct-DNA changes in absorbance and in the position of the band should occur. The experiment was carried out for a constant concentration of free olanzapine (5×10-5 M L-1) with increasing concentrations of ct-DNA (0 to 1×10 -4 molL-1) (Fig. 2). With the addition of ct-DNA, UV spectrum shows a decrease in the peak intensity at 260 nm. The intrinsic binding constant, Kb for olanzapine was calculated from Wolfe– Shimmer Eq. (1), [DNA]/(εa-εf)= [DNA]/(εb-εf) + 1/Kb(εb-εf)

(1)

Where [DNA] is the concentration of DNA in base pairs, εa is the apparent extinction coefficient obtained by calculating Aobs/ [drug] εf corresponds to the extinction coefficient of the drug in its free form and εb refers to the extinction coefficient of the drug in the bound form. Each set of data, when fitted to the above equation, gave a straight line with a slope of 1/ (εb- εf) and a y intercep of 1/Kb(εb – εf) and Kb was determined from the ratio of the slope to intercept (Fig. 2). The calculated Kb value was 2×10 3 M-1 that it compared with groove-binding drugs such as 2- imidazolidinethione and levetiracetam kb = 1.4 × 103 and (4.9 ± 0.2) × 10 3 M-1, respectively [18, 19]. 3.2. CD studies The CD-spectra in the UV range can be used to monitor the conformational transition of DNA [20]. The effect of olanzapine on the conformation of secondary structure of DNA is studied by keeping the concentration of DNA constant (5×10-5 M) and varying the concentration of olanzapine (ri = 0.0 0.6 0.8 0.9 M) in Tris-HCl buffer at pH 7.4. The CD spectra of DNA in presence and absence of the drug are shown in Fig 4. In the absence of the drug, the CD spectrum is of typical Bform that exhibits a positive band at 277 nm and a negative ban at 245 nm as shown in Fig 3. Upon 6

the incremental addition of olanzapine to DNA, the intensity of both the positive and negative bands increased. The changes in the CD spectra in the presence of the drug show stabilization of the righthanded B form of ct-DNA [21]. 3.3. Viscosity measurements Viscometry is an effective tool to determine the mechanism of action of a drug at the ct-DNA molecular level. A classical intercalation binding requires the space of adjacent base pairs to be large enough to accommodate the bound small molecules and to elongate the double helix, resulting in an increase of ct-DNA viscosity [22]. In contrast, there is little effect on the viscosity of DNA if electrostatic or groove binding occurs in the binding process [23]. The ct-DNA viscosity experiments performed at 25 . Increasing amounts of olanzapine (ri = 0–1.8.) were used to evaluate viscosity η of the ct-DNA. The values of relative specific viscosity (η/ η 0)1/3 versus R (R= [drug]/[ct-DNA]) were plotted (Fig. 4). Little changes on the viscosity of ct-DNA showed that the drug bound to ct-DNA by groove binding such as paeoniflorin and sinafloxacin [24]. 3.4. Competitive binding studies by fluorescence studies No luminescence was observed for olanzapine upon excitation either in aqueous solution or in the presence of ct-DNA. To investigate the mode of olanzapine binding to DNA, a competitive binding experiment was performed. Hoechst 33258 binds strongly to the minor groove of double-stranded B-DNA with specificity for AT-rich sequences [25]. The interaction of Hoechst 33258with DNA in Tris–HCl (pH 7.4) was characterized by the fluorescence spectra (Fig. 6). The fluorescence of Hoechst increases in the presence of DNA due to its higher planarity in the grooves of the host macromolecule as well as its protection from collisions with solvent [26]. When olanzapine is added into the solution of DNA–Hoechst complex, the decrease in fluorescence intensity is shown in Fig. 5. There are three possible reasons considering the decrease of the fluorescence intensity for DNA– Hoechst system upon addition of olanzapine. Firstly, the binding

7

between Hoechst and olanzapine might occur and the intensity of DNA– Hoechst complex decreases. Secondly, olanzapine competes against Hoechst in binding with DNA and excludes the groove Hoechst from the duplex, so the concentration of Hoechst binding with DNA reduces. Thirdly, olanzapine binding with DNA– Hoechst forms a new non-fluorescence complex DNA– Hoechst –Olanzapine, which causes the fluorescence quenching of DNA– Hoechst. As shown Fig 6 it is found when olanzapine was added to Hoechst does not react with it. So, the first reason is not true of interpreting the decrease of DNA– Hoechst. The second reason above mentioned seems to be more reasonable, namely with the addition of olanzapine to a solution of DNA– Hoechst complex, some hoechst molecules were released into solution after an exchange with the olanzapine, and this resulted in fluorescence quenching. This supported the view that olanzapine could interact as a minor groove binder. Quenching can occur by different mechanisms, which are usually classified as dynamic and static quenching. Dynamic quenching refers to a process in which the fluorophore and the quencher come into contact during the transient existence of the exited state, while static quenching refers to fluorophore–quencher complex formation. In general, dynamic and static quenching can be distinguished by their differing dependence on temperature and excited-state lifetime. Since in both cases the fluorescence intensity is related to the concentration of the quencher, the quenched fluorophore can serve as an indicator for the quenching agent [27]. In a similar spectrofluorimetric experiment, methylene blue (MB) was added to DNA solution. Phenothiazine dyes, such as methylene blue that can interact with DNA by intercalation have been used in several spectroscopic studies. Upon binding to DNA the fluorescence probe is efficiently quenched by the DNA bases with no apparent shifts in the emission maximum. This emissionquenching phenomenon also reflects the changes in the excited-state electronic structure in consequence of the electronic interactions in the MB–DNA complex. The emission-quenching phenomenon, the hypochromic and red-shift effects in the absorption spectra fit the intercalative mode of binding of MB to DNA [28]. Fig. 7 shows the fluorescence emission spectra of MB with 8

and without ct-DNA and the effect of the addition of olanzapine to MB bound ct-DNA. There is no significant displacement of MB by olanzapine reflecting the absence of an intercalative mode of binding. As there is evidence of complex formation from other experiments, a groove-binding mode could be ascribed to it. Other experimental studies as well as DNA ligand docking have also substantiated this. Fluorescence quenching is described by the Stern–Volmer equation (Eq. (2)): F0/F= 1+Kq τ0 [Q] = 1+ Ksv [Q]

(2)

Where F0 and F represent the fluorescence intensities in the absence and in the presence of quencher, respectively. Kq is the fluorophore quenching rate constant, Ksv is quenching constant, is the lifetime of the fluorophore in the absence of a quencher (

= 10-8) and Q is the

concentration of quencher [29]. Dynamic and static quenching can be distinguished by their different dependence on temperature [30]. Kq, which equals KSV/τ0 is the apparent bimolecular quenching rate constant. For dynamic quenching, the maximum scattering collisional quenching constant of various quenchers is 2×1010 L mol-1 s -1. KSV and Kq obtained from Stern–Volmer equation at different temperatures are presented in Table 1. It is seen that KSV and Kq increased as temperature decreased, indicating that the mechanism of the quenching may be a static quenching. However, Kq is much larger than 2×1010 L mol

-1

s-1, suggesting that the quenching process be a

static quenching [31]. In our experiments, with the temperature increasing from 288 K to 310K, the Stern–Volmer constant (Ksv) decreased from 2.723 ×10 3 M-1 to 1.98 ×103M-1 for olanzapine. These results indicate that the probable quenching mechanism of olanzapine by ct-DNA involves static quenching, because Ksv is decreased with increasing temperature [18].

3.4.1. Equilibrium binding titration

9

The binding constant (Kf) and the binding stoichiometry (n) for the complex formation between olanzapine and ct-DNA were measured according to the method described by Hu et al Eq. (3) [32] Log (F0– F/F) = Log Kf + n Log [Q]

(3)

Here, F0 and F are the fluorescence intensities of the fluorophore in the absence and presence of different concentrations of quencher, respectively. In the present study, the binding constants of the drug were obtained at various temperatures. The values of Kf and n at 298 were 2×10 3 l mol-1 and 1.02, respectively (Table 1). The value of Kf for olanzapine at room temperature is comparable to N,N-Bis(3β-acetoxy-5α-cholest-6-yl-idene)hydrazine (4.7× 10 3 M−1) and resistomycin (3.23× 103 M−1) [33] which binds to DNA in a groove binding mode [34]. 3.4.2. Thermodynamic studies To obtain a detailed view of the interaction between olanzapine and DNA, a powerful approach is to parse the free energy of the binding reaction into its component terms. The four classes of noncovalent interactions that can play roles in the binding of drug molecules to biomolecules are: hydrogen bonds, van der Waals forces, electrostatic interactions and hydrophobic bond interactions [35]. Consequently, in this study, the formation constant of olanzapine–DNA complex formation is evaluated at three different temperatures, allowing for the determination of thermodynamic parameters such as enthalpy (∆H) and entropy (∆S) by the van’t Hoff equation: LNK = - ∆H/RT + ∆S/R

(4)

∆G = ∆H - T∆S

(5)

Here K is the binding constant at the corresponding temperature and R is gas constant. ∆H and ∆S were obtained from the slope and intercept of the linear plot (Eq. (4)) based on lnK versus 1/T (Fig.8).The free energy change( ∆G) were calculated from Eq. 5 and are listed in Table 1. Ross and coworkers reported that when ∆H < 0 or ∆H and ∆S > 0, the electrostatic force dominates the

10

interaction; when ∆H < 0 and ∆S < 0, van der Waals interactions or hydrogen bonds dominate the reaction and when ∆H > 0 and ∆S > 0, hydrophobic interactions dominate the binding process [36,37]. When we applied this analysis to the binding system of olanzapine and ct-DNA, we found that ∆H < 0 and ∆S < 0. Therefore, van der Waals interactions or hydrogen bonds are the main forces involved in the binding of olanzapine to ct-DNA. 3.5. Molecular docking studies on the interaction mechanism of olanzapine with DNA Molecular docking technique is an attractive scaffold to understand the drug-DNA interactions for the rational drug design and discovery, and also in the mechanistic study by placing a small molecule into the binding site of the target specific region of the DNA mainly in a non-covalent fashion, although covalent bond may also be constituted with reactive ligand and to predict the correct binding mode and binding affinities [38]. Molecular models were built to discuss the binding modes by docking using AutoDock program for the interactions of olanzapine molecule with multiple ct-DNA fragments. The structure of each drug was drawn and subjected to energy optimization [39]. These were then imported to ct-DNA molecule for docking purpose. The resulting drug–DNA complex was used for calculating the energy parameters which can substantiate the spectroscopic results [40]. It is well known that the interactions of chemical species with the groove binding of B-DNA differ from those occurring in the major groove, both in terms of electrostatic potential and steric effects, because of the narrow shape of the former. Small molecules interact with the minor groove, while large molecules tend to recognize the major groove binding site [41]. From the docking calculation, the conformer with minimum binding energy is picked up from the 9 minimum energy (root mean square deviation; rmsd = 0) conformers from the 100 runs. The best possible conformation of the olanzapine –DNA complex shown in Fig. 8 clearly indicated that the minor groove binding was the most probable interaction mode between olanzapine and DNA, which was agreeable with the experiment results. From the results, we could find that the DNA residues with ID of T8, T19, A17 and A18, C9 and G10 played a major role in the binding 11

site with the optimal energy. In this work, the hydrogen bond as long as 2.17 A ˚ formed between the O atom in phosphate group of G10 on chain B (DT20B) from DNA and the H atom of amine from olanzapine (Fig. 9). According this result was coincident with the conclusion that olanzapine interacts with ct DNA through the minor groove binding. 4. Conclusion In this paper , we have studied the binding of olanzapine with ct-DNA. Results suggest that the drug binds to ct-DNA via minor groove binding mode. The interaction occurrence is supported by the following findings : (1) The intrinsic binding constant (Kb = 2×103 M-1) is more in keeping with groove binding with DNA.(2) little effect on the viscosity of ct-DNA (3) Decrease in the fluorescence intensity of the Hoechst-DNA adding the olanzapine. The thermodynamic parameters (∆H < 0 and ∆S < 0) indicated that hydrogen bond and van der Waals play main roles in the binding of the drug to ct-DNA. (4) Optimal results of docking were all in the AT-rich and GC-rich region. Thus, the olanzapine molecule was relatively easy binds with the minor groove of the duplex ctDNA with a certain preference of the binding of the AT, GC region. As DNA groove binders constitute an important class of derivatives in anticancer therapy [42].

References [1] NA. Moore, DC. Calligaro, Wong DT, Bymaster FP, Tye NC, Curr Opin Investig Drugs 2 (1993) 281-293. [2] F. P. Bymaster, D. Calligaro, J. Falcone et al, Neuropsychopharmacology14 (1996) 87–96. [3] F. P. Bymaster, JF. Falcone, D. Bauzon et al, Eur J Pharmacol 430 (2001) 341–349. [4] J.B. Chaires, Biopolymer (Nucleic Acid Sci.) 44 (1998) 201–215. 12

[5] K. Naing, M. Takahashi, M. Taniguchi, A. Yamagishi, Bull. Chem. Soc. Jpn. 67 (1994) 2424. [6] A.M. Pyl, J.P. Rehman, R. Meshoyrer, C.V. Kumar, N.J. Turro, J.K. Barton, J. Am. Chem. Soc. 111 (1989) 3051–3058. [7] N. Shahabadi , A. Fatahi, Molecular Structure 970 (2010) 90–95. [8] T. Biver, F. Secco, M.R. Tine, M. Venturini, J. Inorg. Biochem. 98 (2004) 33–40. [9] C. Liu, J. Zhou, H. Xu, J. Inorg. Biochem. 71 (1998) 1–6. [10] S. Mahadevan, M. Palaniandavar, Inorg. Chim. Acta 254 (1997) 291–302. [11] C.A. Mitsopoulou, C.E. Dagas, C. Makedonas, Inorg. Chim. Acta 361 (2008) 1973–1982. [12] J. Sambrook, E.F. Fritsche, and T. Maniatis, Molecular Cloning: A Laboratory Manual, third ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. [13] G.D. Liu, J.P. Liao, Y.Z. Fang, S.S. Fang, G.L. Huang, R.Q. Sheng, J. Yu, Anal. Sci. 18 (2002) 391–395. [14] V.A. Bloomfield, D.M. Crothers, I. Tioco, New York (1974) 432–434. [15] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, J. Comput. Chem. 30 (2009) 2785–2791. [16] G.M. Morris, R. Huey, A.J. Olson, Curr. Protoc. Bioinf. 8 (2008) 14.1–8.14. [17] W.L. DeLano, San Carlos, CA, USA, 2004. [18] N. Shahabadi, S. Hadidi, Spectrochim. Acta A 96 (2012) 278–283. [19] F. Ahmadi, A.A. Alizadeh, F. Bakhshandeh-Saraskanrood, B. Jafari, M. Khodadadian, Toxicol. 48 (2010) 29–30. [20] Y. Zhou, Y. Li, Colloids Surf. A: Physicochem. Eng. Aspects 233 (2004) 129–135. 13

[21] N. Shahabadi , S. Kashanian , M. Khosravi , M. Mahdavi, Met Chem 35 (2010) 699–705 [22] L.S. Lerman, J. Mol. Biol. 3 (1961) 18–30. [23] G. Zhang, P. Fu, J. Pan, J. Lumin. 134 (2013) 303–307. [24] Y. Fei, G. Lu, G. Fan, Y. Wu, Anal. Sci 25 (2009) 1335. [25] R. Kakkar, R. Garg, Suruchi, J. Mol. Struct. 579 (2002) 109–113. [26] Y. Guan, W. Zhou, X. Yao, Anal. Chim. Acta 570 (2006) 21–28. [27] M.R. Eftink, C.A. Ghiron, Anal. Biochem. 114 (1981) 199–227. [28] Y. Sun, S. Bi, D. Song, C. Qiao, D. Mu, H. Zhang, Sens. Actuators, B 129 (2008) 799–810. [29] F.L. Cui, J. Fan, J.P. Li, Z.D. Hu, Bioorg. Med. Chem. 12 (2004) 151–157. [30] H. Chen, S. S. Ahsan, M. B. Santiago-Berrios, H. D. Abruna and W. W. Webb, J. Am. Chem. Soc 132 ( 2010) 7244–7245. [31] M. Jiang, M.X. Xie, D. Zheng, Y. Liu, X.Y. Li, X. Chen, J. Mol. Struct. 692 (2004) 71–80 [32] R. Vijayabharathia, P. Sathyadevi, P. Krishnamoorthy, D. Senthilraja, P. Brunthadevi, S. Sathyabama and V. BrindhaPriyadarisini, Spectrochim. Acta A 89 (2012) 294– 300. [33] Z. Tabassum, M. Muddassir, O. Sulaiman and F. Arjmand, J. Lumin. 132 (2012) 2178–2181. [34] I. Haq, Arch. Biochem. Biophys. 403 (2002) 1–15 [35] F. Ahmadi, A.A. Alizadeh, N. Shahabadi, M. Rahimi-nasrabadi, Spectrochim. Acta A 79 (2011) 1466–1474. [36] N. Shahabadi, A. Fatahi, J. Mol. Struct. 970 (2010) 90–95. 14

[37] T.G. Appleton, J.R. Hall, J. Inorg. Chem. 9 (1970) 1807–1813. [38] I. Haq and J. Ladbury, J. Mol. Recog. 13 (2000) 188–197. [39] E. M. Proudfoot, J. P. Mackay and P. Karuso, BioChem.: Indian J. 40 ( 2001) 4867-4878. [40] F. Arjmand, S. Parveen, M. Afzal, L. Toupet and T. B. Hadda, Euro. J. Med. Chem. 49 (2012) 141-150. [41] R. Hajian, M. Tavakol, J. Chem. 9 (1) (2012) 471–480. [42] P.G. Baraldi, A. Bovero, F. Fruttarolo, D. Preti, M.A. Tabrizi, M.G. Pavani, R. Romagnoli, Med.Res. Rev. 24 (2004) 475–528.

15

Figure captions Fig. 1. Chemical structure of olanzapine Fig. 2. Absorption spectra of olanzapine (5×10-5 M) in the absence and presence of increasing amounts of CT-DNA: ri = [DNA] / [Drug]: 0.0, 0.3, 0.5, 0.8, 1/2, 1.7, 2. Inset: Plot of [DNA/ (εa– εf)] vs [DNA] Fig. 3. CD spectra of DNA (5×10-5 M) in 10 mM Tris-HCl buffer, in the presence of increasing amounts of the drug (ri = [Drug]/[DNA] =0.0, 0.6, 0.7, 0.9) Fig. 4. Effect of increasing concentration of drug on the relative viscosity of ct-DNA at 25 ºC Fig. 5. The fluorescent spectral characteristics of drug–DNA at 283 K. The DNA concentration: 2.5×10-4 molL-1 in pH 7.4 Tris-buffer. Drug concentrations: 0.00 , 0.57, 0.76,3.7, 4.7, 7.8, 9, 10, 14, 20, 24, 28,and 30×10 -5 mol L-1. Fig. 6. The fluorescent spectral characteristics of drug–Hoechst at 283 K. Drug concentrations: 0.00 , 0.57, 0.76,3.7, 4.7, 7.8, 9, 10, 14, 20, 24, 28,and 30×10-5 mol L-1. Fig. 7. Fluorescence spectra of the competition between olanzapine and MB. C Olanzapine = 24.6, 73.3, 1.2, 1.69 2.3 2.8 3.1 and 3.4× CMB = 5×

mol

and C DNA = 1.5×

mol

mol

,

at 298K.

Fig. 8. The plot of log Kf versus 1/T Fig. 9. Molecular docked model of olanzapine showing groove binding with DNA dodecamer duplex of sequenced (CGCGAATTCGCG)2 (PDB ID): 1BNA

16

Table 1. The quenching constants (Ksv, Kq), binding constants (Kf), number of binding sites (n) and relative thermodynamic parameters for the binding of olanzapine to ct-DNA

T (0K)

∆G

∆H

∆S

(kj mol-1)

(kj mol-1)

(j mol-1)

-52.89

-114.89

Ksv (l mol-1) ×10-3

Kq(lmol-1) ×10-11

Kf(lmol-1) ×10-3

n

288

2.72

2.72

3.49

1.03

-19.80

298

2.13

2.13

2.28

1.01

-18.65

310

1.98

1.98

735.0

0.87

-17.27

Fig. 1

17

Fig. 2

Fig. 3

18

Fig. 4

Fig. 5

19

Fig. 6

Hoechst Hoechstolanzapine

Fig 7:

Fig. 8 20

Fig. 9

21

Graphical abstract

23

Research Highlights: •

Through the site marker competitive experiment, Olanzapine was confirmed to be located in minor groove



The thermodynamic parameters indicated that the hydrogen bonding and weak van der Waals force played a major role in the interaction



The experimental results were in agreement with the results obtained via a molecular docking study.

24

Spectroscopic and molecular docking studies on the interaction of the drug olanzapine with calf thymus DNA.

The present study investigated the binding interaction between olanzapine and calf thymus DNA (ct-DNA) using emission, absorption, circular dichroism,...
716KB Sizes 0 Downloads 8 Views