Accepted Manuscript Spectroscopic and computational studies on the interaction of DNA with pregabalin drug Nahid Shahabadi, Sara Amiri PII: DOI: Reference:

S1386-1425(14)01601-1 http://dx.doi.org/10.1016/j.saa.2014.10.104 SAA 12919

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

25 July 2014 14 October 2014 23 October 2014

Please cite this article as: N. Shahabadi, S. Amiri, Spectroscopic and computational studies on the interaction of DNA with pregabalin drug, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.10.104

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 computational studies on the interaction of DNA with pregabalin drug Nahid Shahabadi1*,Sara Amiri1

1

Department of Chemistry, Faculty of Science, Razi University, Kermanshah, Iran.

* Corresponding author: Tel / Fax: +98-83-34274559 E-mail: [email protected]

1

Abstract The interaction of the drug pregabalin (S-3-(aminomethyl)-5-methylhexanoic acid) with CTDNA was studied by using fluorescence spectroscopy, UV-Vis, CD, molecular docking study and viscometery. The fluorescence and UV absorption spectroscopy indicated that the drug interacted with CT-DNA in a groove binding mode. The binding constant and the number of binding sites were 5.6×104 L mol-1 and 0.96, respectively. The fluorimetric studies showed that the reaction between the drug and CT-DNA is exothermic (∆H = 33.11 KJ mol-1; ∆S = 48.84 J mol-1 K-1). Furthermore, the drug doesn´t induce any changes in DNA viscosity. Circular dichroism spectroscopy (CD) was employed to measure the conformational changes of CT-DNA in the presence of the drug, which verified the groove binding mode. The molecular modeling results illustrated that the drug binds to groove of DNA by relative binding energy of docked structure - 21.9 KJ mol-1. Keywords: Pregabalin, DNA interaction, Molecular docking, Groove binding.

1. Introduction: Pregabalin, ((S)-3-(aminomethyl)-5-methylhexanoic acid) (Fig.1) binds with high affinity to the α2δ subunit of voltage-gated calcium channels and exerts analgesic, anxiolytic, and antiseizure activities. Pregabalin undergoes minimal metabolism in most species, including humans, and has a wide tissue distribution, with concentrations in most tissues similar to that in blood. Renal excretion is the primary route of elimination. Analysis of pregabalin for structural alerts using the in silico predictive systems DEREK and CASE/Multicase suggests that pregabalin contains no biologically relevant molecular features associated with genotoxicity or carcinogenicity [1]. Structurally, pregabalin is related to the endogenous

2

amino acid L-leucine and to the naturally occurring inhibitory neurotransmitter γ-amino butyric acid (GABA) (Fig.2). According to in vivo studies, oxaliplatin fights carcinoma of the colon through nontargeted cytotoxic effects. Like other platinum compounds, its cytotoxicity is thought to result from inhibition of DNA synthesis in cancer cells. Pregabalin significantly reduced the severity of oxaliplatin-induced sensory neuropathy. Being more potent than gabapentin, pregabalin achieved efficacy at lower doses and should lead to fewer dose-related side effects, although this remains to be established in a head-to-head trial. The acute hyper excitability syndrome caused by oxaliplatin has also been treated with partial success with the use of anticonvulsant agents like carbamazepine and gabapentin. Recent studies on B6C3F1 and CD-1 mice and two separate studies in Wistar rats along two years showed that pregabalin treatment was associated with an increased incidence of hemangiosarcoma primarily in liver, spleen, and the incidence of hemangiosarcoma was higher in B6C3F1 mice than in CD-1 mice, consistent with its spontaneous incidence. Pregabalin did not increase the incidence of any other tumor type in rats and was not genotoxic, based on an extensive battery of in vivo and in vitro tests in bacterial and mammalian systems. Therefore, pregabalin treatment selectively increased the incidence of a single tumor type (hemangiosarcoma) via a nongenotoxic mechanism in mice, a species predisposed to development of this tumor type. Hem angiosarcomas from affected mice were genotypically distinct from their human counterparts, further suggesting a species-specific mechanism of origin. According to the results of the previous studies [2] we try to do more experiments about the mechanism of the action of this drug with DNA.

3

The aim of this study is to find the mode of the interaction between pregabaline and DNA. This will help us to know about the mechanism of damaging DNA in the presence of this drug. The advantages of the proposed method involve simplicity and cheap devices.

Experimental

2. Materials and methods 2.1. Materials The highly polymerized calf thymus DNA (CT-DNA), Hoechst 33258 and Tris–HCl were purchased from Sigma Co. Methylene blue was purchased from Merck. All solutions were prepared using double distilled water. Tris–HCl buffer solution was prepared from (Tris–(hydroxymethyl)-amino-methane– hydrogen chloride) and pH was adjusted to 7.4. The stock solution of DNA was prepared by dissolving of DNA in 50 mM of the Tris–HCl buffer at pH 7.4 and dialysing exhaustively against the same buffer for 24 h and used within 5 days. A solution of CT-DNA gave a ratio of UV absorbance at 260 and 280 nm more than 1.8, indicating that DNA was sufficiently free from protein [3]. The concentration of the nucleotide was determined by UV absorption spectroscopy using the molar absorption coefficient (ε = 6600 M -1 cm-1) at 260 nm. The stock solution was stored at 4 ◦C. Stock solution of pregabalin was prepared by dissolving 0.6 mg of the drug in 4.0 mL of Tris–HCl buffer 10 mM (final concentration = 10-3 mol1. L-1).

2.2. Instrumentation 4

The UV–Vis spectra for DNA–drug interactions were obtained using an Agilent 8453 spectrophotometer. Solutions of DNA and pregabalin were scanned in a 1 cm quartz cuvette. Absorption experiments were carried out by keeping the concentration of DNA constant (1.6×10 -4 M) while varying the drug concentration from 7.1×10 -6 to 2.2×10 -4 M (ri = [complex] / [DNA] = 0.044 - 1.37). Absorbance values were recorded after each successive addition of DNA solution and equilibration (ca. 24 h). Fluorescence measurements were carried out with a JASCO spectrofluorimeter (FP 6200). The competitive interaction between the Hoechst 33258 and the pregabalin drug with DNA was carried out as follows: fixed amounts of the Hoechst 33258 and DNA were titrated with increasing amounts of pregabalin solution. All fluorescence measurements were performed on a JASCO spectrofluorimeter (FP6200) using a quartz cuvette of 1 cm path length by keeping the concentration of the DNA constant (2.4 × 10

-5

mol1. L-1) while varying the drug

concentration from 3.9×10 -7 to 3 × 10 -4 M at three different temperatures (283, 295, and 310 K). Viscometery is an effective tool to determine the mechanism of action of a drug at the CTDNA molecular level. Viscosity measurements were made using a viscosimeter (SCHOT AVS 450) maintained at 25°C

0.5° C in a constant temperature bath. The DNA

concentration was fixed at 3×10-3 M. Flow time was measured with a digital stopwatch; the mean value of three replicated measurements was applied to evaluate the viscosity (η) of the samples. The values of 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 1/R (R = [DNA]/[complex]) [4].

5

CD measurements were recorded on a JASCO (J-810) spectropolarimeter, keeping the concentration of DNA constant while varying the drug concentration (ri = [complex]/ [DNA] =0.0,0.1,0.2).

2.3. Molecular docking study MGL tools 1.5.4 with AutoGrid4 and AutoDock4 [5-6] were used to set up and perform blind docking calculations between the drug and DNA sequence. Drug structure obtained from the Drug Bank (Drug ID: DB00718) 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, 72×96×126 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 [7].

3. Results and Discussion 3.1. Electronic absorption spectra UV–Vis absorption measurement is a simple but effective method in detecting complex formation. In general, when a small molecule interacts with DNA and forms a new complex, changes in absorbance and in the position of the band should occur [8]. The experiment was carried out for a constant concentration of DNA with increasing concentrations of the drug that is shown in Fig. 3. So, with increasing the concentration of pregabalin, DNA spectrum 6

shows decrease in the peak intensity at 260 nm. In addition, pregabalin is a small molecule with the low bonding size, therefore the minor groove binding is preferable than the major. It is reported that groove binding molecules typically have unfused aromatic ring systems linked by bonds with torsional freedom for the molecules to adopt appropriate conformation that attentively matches the helical turn of DNA grooves [9]. The values of the binding constant, Kb, were obtained from the DNA absorption at 260 nm according to the methods published in the literature [10], where the bindings of various ligands to hemoglobin were described. For weak binding affinities, the data were treated using linear reciprocal plots based on the Eq. 1 [11].

1/(A-A0) =1/(A∞-A0) + 1/K(A∞-A0) . 1/[Prg]

(1)

where A0 is the absorbance of DNA at 260 nm in the absence of pregabalin, A∞ is the final absorbance of the pregabalin–DNA and A is the recorded absorbance at different drug concentrations. The double reciprocal plot of 1/(A–A0) versus 1/[drug] is linear

and the

binding constant (K) can be estimated to be 5.6×104 from the ratio of the intercept to the slope .This Kb value is similar to the binding constant ,Kb, for the metformin and DNA (8.3 × 104 M-1). The Kb value obtained for the drug compares well with that of the well-established groove binding agent spermine [12].

3.2. Fluorescence spectra One approach to study the binding mechanism between CT- DNA and a drug is fluorescence quenching technique. Fluorescence quenching refers to any process which decreases the fluorescence intensity of a sample [13].

7

Quenching can arise by different mechanisms, which are usually classified as dynamic and static quenching. Dynamic and static quenching can be distinguished by their different dependence on temperature. In fact, dynamic quenching refers to a process in which the fluorophore and the quencher come into contact during the transient existence of the exited state, but static quenching refers to fluorophore-quencher complex formation. For the dynamic quenching, higher temperatures will result in faster diffusion and larger amounts of collisional quenching, hence the quenching constant values will increase with increasing temperature, but the reverse effect would be observed for static quenching. To clarify quenching mechanism, fluorescence quenching spectra were measured at different temperatures [14]. The interaction of Hoechst 33258 with DNA in Tris–HCl (pH 7.4) was characterized by the fluorescence spectra. Fluorescence emission spectra of DNA- Hoechst are significantly enhanced by increasing the DNA concentration [15]. The fluorescence intensity decreases by adding the pregabalin drug into the solution of DNA–Hoechst complex (Fig. 4). Fluorescence quenching is described by the Stern–Volmer equation (Eq. 2): F0/F = 1+ Kqτ0[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, τ0 is the lifetime of the fluorophore in the absence of a quencher (τ0 = 10 -8), and [Q] is the concentration of quencher [16]. The results in Table 1 indicate that the probable quenching mechanism of DNA

by the drug involves static quenching, because Ksv is

decreased with increasing temperature [17].

3.2.1. Measurement of Kf

8

The binding constant (Kf) and the binding stoichiometry (n) for the complex formation between pregabalin and DNA were measured using the Eq. 3 [18].

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 in the presence of different concentrations of [Q], respectively. The values of Kf and n are shown in Table 1. The value of Kf for pregabalin at room temperature is comparable to resistomycin (3.23× 103 M-1) [19], N,N-Bis(3β-acetoxy-5α-cholest-6-yl-idene)hydrazine (4.7× 103 M−1) [19] which binds to DNA in an groove binding mode [20]. The interaction forces between a drug and biomolecule may involve hydrophobic forces, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc [21]. According to the data of enthalpy changes (∆H) and entropy changes (∆S), the model of interaction between the drug and biomolecule can be concluded [22] : (1) ∆H > 0 and ∆S > 0, hydrophobic forces; (2) ∆H < 0 and ∆S < 0, van der Waals interactions and hydrogen bonds; (3) ∆H < 0 and ∆S > 0, electrostatic interactions [23]. When there is little change of temperature, the enthalpy change (∆H) can be seen as a constant, and then its value and that of entropy changes (∆S) can be determined from the van’t Hoff equation:

LNK = - ∆H/RT+∆S/R

(4)

∆G = ∆H-T∆S

(5)

where K is the binding constant at the corresponding temperature and R is gas constant. The values of ∆H and ∆S were obtained from the slope and intercept of the linear plot (Eq. 4) based on lnK versus 1/T. The free energy change (∆G) was estimated from Eq. 5. The values 9

of ∆H, ∆S and ∆G between pregabalin and DNA are listed in Table 1. It can be seen that the negative value of ∆G revealed the interaction process is spontaneous, while the negative ∆H and ∆S values indicated that hydrogen bond and van der Waals play main roles in the binding of drug to DNA. According to the thermodynamic data, the formation of the DNA– pregabalin complex is enthalpy favored while it is entropy disfavored. The complex formation results in a more ordered state, possibly due to the freezing of the motional freedom of both the pregabalin and DNA molecules.

3.3. Viscosity Measurements Viscometery is an effective tool to determine the mechanism of action of a drug at the CT-DNA molecular level. The hydrodynamic measurements are sensitive to the length change of DNA in absence and presence of foreign molecules. A classical intercalation model demands that the DNA helix must lengthen as base pairs are separated to accommodate the binding ligand, leading to increase in DNA viscosity [24]. In contrast, a partial, non-classical intercalation of molecule could bend (or kink) the DNA helix, reducing its length and, concomitantly, its viscosity. In addition, molecules that bind exclusively in the DNA grooves by partial and/or nonclassical intercalation, under the same conditions, typically cause less pronounced (positive or negative) or no change in DNA solution viscosity [25]. The values of relative specific viscosity (η/η0)1/3 versus 1/R (R = [DNA]/[complex]), in the absence and in the presence of pregabalin were plotted (Fig. 5) . Little change on the viscosity of CT-DNA showed that the drug bound to CT- DNA by groove binding such as paeoniflorin and sinafloxacin [26-27].

3.4. CD Spectral Studies

10

CD spectroscopy is a useful technique for analyzing interactions between drugs and CTDNA. It is also useful because CD signals are quite sensitive to the mode of DNA interaction with small molecules. Since different DNA structures have different CD spectra, this technique is a powerful procedure to understand the conformational changes of DNA. The CD spectra of DNA in the presence of the drug were illustrated (Fig.6). The CD spectra of DNA with different values of the drug exhibit increases in the positive and in the negative bands. These CD changes represent more stacking of DNA base pairs due to the hydrophobic interaction in groove region and decrease in the helicity of DNA by unwinding it. In particular, B-DNA shows two conservative CD bands in the UV region: a positive band at 280 nm due to base stacking and a negative band at 245 nm because of the right handed helicity. The changes in the CD spectra in the presence of the drug show stabilization of the right-handed B form of CT-DNA [28-29].

3.5. Molecular docking analysis Computer docking techniques play an important role in drug design and elucidation of mechanism. Also, molecular docking technique is an attractive scaffold for 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 [30]. These docking programs, when used prior to experimental screening, can be considered as powerful computational filters to reduce labor and cost needed for the development of effective medicinal compounds. In addition, they can help in better understanding of bioactivity mechanisms [31]. The structure of each drug was drawn and subjected to energy optimization. The resulting drug- DNA complex was used for calculating the energy parameters [32] which can substantiate the spectroscopic results. Small molecules interact 11

with the minor groove, while large molecules tend to recognize the major groove binding site [33]. From the docking calculation, the conformer with minimum binding energy is picked up from the 20 minimum energy (root mean square deviation; rmsd = 0) conformers from the 100 runs [34-35]. The run data for the conformers are listed in Table 2. The initial conformation was taken from one of the lowest binding energy docking conformation (Table 2). The energetically most favorable conformation of the docked pose (Fig.7) revealed that pregabalin binds to minor groove of DNA. From the docking results with the optimal energy, it was found that the drug molecule inserted into the minor groove of DNA fragments and had van der Waals forces with double strands of the duplex DNA. There were the formation of hydrogen bonds between one oxygen atom and two hydrogen atoms of the pregabalin molecule and residues G10 , C11and G12 of the DNA fragment with bond lengths of 2.63 A˚ , 1.89 A˚ and 1.89 A˚, respectively. From the docking simulation the observed free energy change of binding (∆G) for the pregabalin-DNA complex is calculated to be -5.24 kcal Mol-1 which is slightly higher than the experimental free energy of binding (-19.2 KJ. mol-1 = - 4.47 kcal mol-1 ) obtained from the fluorescence data. This apparent mismatch in the free energy changes could be due to the exclusion of the solvent and/or rigidity of some other receptor DNA in the molecular docking studies. Furthermore, the binding constant obtained by UV– visible method was correlated with the free binding energy of docked model. Basic formula of binding constant and Gibbs free energy is: ∆G= - RTLnK

(6)

the binding constant obtained by UV–vis (5.6 × 104 L.mol-1) matches to the binding constant calculated by docked drug–DNA model (7.2 × 103 L.mol-1) Hence, it can be concluded that drug–DNA

docked

model

experimental results.

12

is

in

approximate

correlation with our

4. Conclusion In this paper we have studied the binding of CT-DNA with the drug pregabalin. In fact, different instrumental methods were used to finding the interaction mechanism. Pregabalin were examined by absorption, fluorescence, viscosity measurements, CD spectra and docking. The results suggested that the drug binds to DNA via groove binding mode. The interaction occurrence is supported by the following findings: 1. The intrinsic binding constant (Kb = 5.6 × 10 4 M-1) is more in keeping with groove binding with DNA. 2. Decrease in the fluorescence intensity of the CT-DNA in the presence of pregabalin. 3. The thermodynamic parameters (∆H < 0 and ∆S < 0) indicated that hydrogen bond and van der Waals play main roles in the binding of pregabalin to CT-DNA. 4. The drug showed a little effect on the viscosity of CT-DNA. 5. Circular dichroism results showed stabilization of the right-handed B form of CT-DNA. 5. The docking results revealed that groove mechanism is followed by pregabalin to bind with DNA. Thus, the pregabalin molecule was relatively bound with the minor groove of the duplex CTDNA. As DNA minor groove binders constitute an important class of derivatives in anticancer therapy [36].

References [1] S.K Duddy, R.F Parker, M.R Bleavins, A.W Gough, P.E Rowse, S Gorospe, L.A Dethloff and de la Iglesia, F. A. (1999b) .Toxicol. Appl. Pharm. 156, 106–112. [2] toxicological sciences.,2012, 128(1) , 9–21. [3] G.D. Liu, J.P. Liao, Y.Z. Fang, S.S. Huang, G.L. Sheng, R.Q. Yu, J. Anal. Sci ., 2002 , 13

18 , 391–395. [4] V.A. Bloomfield, D.M. Crothers, I. Tioco, New York .,1974, 432–434. 5 G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K. Belew, D.S. Goodsell, A.J. Olson, J. Comput. Chem.,2009, 30 , 2785–2791. [6] G.M. Morris, R. Huey, A.J. Olson, Protoc. Bioinf., 2008, 8 , 14.1–8.14. [7] W.L. DeLano , DeLano Scientific, SanCarlos, CA, USA., 2004. [8] Y. Fei, G. Lu, G. Fan, Y. Wu . Sci. , 2009, 25 , 1335. [9] Z. Q. Liu, Y. T. Li, Z. Y. Wu and S. F. Zhang, Inorg. Chim. Acta, 2009, 362, 71–77. [10] J.J. Stephanos, J. Inorg. Biochem. ,1996 ,62 , 155–169. [11] R. Marty, N.Ch. Nsoukpoe-Kossi, D. Charbonneau, C.M. Weinert, L. Kreplak, H.A. Tajmir-Riahi, Nucleic Acids Res., 2009 ,37 , 849–857. [12] L. Strekowski, D.B. Harden, R.L. Wydra, K.D. Stewart, W.D. Wilson, J. Mol. Recognit.,1989 ,2 , 158. [13] Y. Wang, J. Lu, H. Yang, Soc. Ethiop., 2009, 23 (1) , 113–115. [14] Y. Zhang, G. Zhang, Y. Li, Y. Hu, J. Agric. Food Chem. , 2013 ,61, 2638–2642. [15] Y. Guan, W. Zhou, X. Yao, Anal. Chim. Acta ., 2006 ,570 , 21–28. [16] Y. Sun, S. Bi, D. Song, C. Qiao, D. Mu, H. Zhang, Actuat., 2008, B129, 799–810. [17] P.B. Kandagal, S.M.T. Shaikh, D.H. Manjunatha, J. Seetharamappa, B.S. Nagaralli, J. Photochem. Photobiol. , 2007 , 189,121–127. [18] M. Jiang, M.X. Xie, D. Zheng, Y. Liu, X.Y. Li, X. Chen, J. Mol. Struct. ,2004 ,692, 71– 80. [19] R. Vijayabharathia, P. Sathyadevi, P. Krishnamoorthy, D. Senthilraja, P. Brunthadevi, S. Sathyabama and V. BrindhaPriyadarisini, Spectrochim. Acta, A, 2012, 89, 294– 300.

14

[20] R. Vijayabharathia, P. Sathyadevi, P. Krishnamoorthy, D. Senthilraja, P. Brunthadevi, S. Sathyabama and V. BrindhaPriyadarisini, Spectrochim. Acta, A, 2012, 89, 294– 300. [21] Z. Tabassum, M. Muddassir, O. Sulaiman and F. Arjmand, J. Lumin., 2012, 132, 2178– 2181. [22] D.B. Nike, P.N. Moorty, K.I. Priyadarsini, Chem. Phys. Lett. , 1990 ,168 , 533–538. [23] N. Shahabadi, A. Fatahi, J. Mol. Struct. ,2010, 970, 90–95. [24] J.B. Chaires, Biopolymer (Nucleic Acid Sci)., 1998, 44,201–215. [25] S. Satyanarayana, J.C. Dabrowiak, J.B. Chaires, Biochemistry ,1992, 31,9319– 9324. [26] G. Zhang, P. Fu, J. Pan, J. Lumin .,2013,134, 303–307. [27] Y. Fei, G. Lu, G. Fan, Y. Wu, Sci,.2009,25, 1335. [28] N. Shahabadi, S. Kashanian, M. Khosravi, M. Mahdavi, Trans. Met. Chem.,2010,35, 699–705. [29] E. Froehlich, J.S. Mandeville, C.M. Weinert, L. Kreplak, H.A. Tajmir-Riahi, Biomacromolecules ,2011, 12 (2) , 511–517. [30] I. Haq and J. Ladbury, J. Mol. Recog., 2000, 13, 188–197. [31] R. Hajian, M. Tavakol, J. Chem., 2012, 9 (1) ,471–480. [32] R. Corradini, S. Sforza, T. Tedeshi, R. Marchelli, Chirality ,2007, 19 ,269–294. [33] R. Hajian, M. Tavakol ,J. Chem., 2012, 9 (1) ,471–480. [34] S. Neelam, M. Gokara, B. Sudhamalla, D.G. Amooru, R.J. Subramanyam, J. Phys. Chem. ,2010, B 114,3005–3012. [35] B. Sudhamalla, M. Gokara, N. Ahalawat, D.G. Amooru, R.J. Subramanyam, J. Phys. Chem. ,2010, B 114 , 9054–9062. 15

[36] P.G. Baraldi, A. Bovero, F. Fruttarolo, D. Preti, M.A. Tabrizi, M.G. Pavani, R. Romagnoli, , Med. Res. Rev. ,2004, 24 , 475–528.

16

Table 1. KSV

,

Kq , binding constants (Kf), number of binding sites (n) and relative

thermodynamic parameters for the binding of pregabalin to CT-DNA. T(0K)

n

Kf (M-1)

Ksv×103 (M-1)

Kq×1011(M-1)

283

1.0951 3468.17

295 310

∆G (KJ mol-1)

∆H (KJ mol-1)

∆S(J mol-1K-1)

1.62

1.62

-19.2

-

-

1.0457 2240.78

1.59

1.59

-18.7

- 33.11

- 48.84

1.0224 1024

0.93

0.97

-17.9

-

-

Table 2. Docking summary of DNA with pregabalin drug by the AutoDock Program Generating Different Ligand Conformers Using a Lamarkian GA. rank

run

Binding energy

a

Ki

(kcalM-1)

Ligand

Ka(M-1)

efficiency

Cluster

Reference

rmsd

rmsd

1

31

-5.24

144.07 uM

-0.48

7.2×10 3

0

29.5

2

39

-5.05

197.91 uM

-0.46

5.1×103

0

29.46

3

30

-4.8

301.72 uM

-0.44

4×103

0

29.25

4

49

-4.3

706.86 uM

-0.39

1.1×103

0

26.18

5

37

-4.22

809.14 uM

-0.38

1.7×103

0

29.26

6

29

-4

1.16 mM

-0.36

8.3×102

0

28

7

5

-4

981.9 uM

-0.37

8.3×102

0

31.42

8

43

-3.95

1.26 mM

-0.36

9×102

0

30.74

9

11

-3.69

1.96 mM

-0.34

6×102

0

27.76

10

10

-3.6

4.86 mM

-0.29

4.4×102

0

30.21

11

12

-3.59

2.33 mM

-0.33

5×102

0

25.84

17

12

18

-3.59

2.33 mM

-0.33

5×102

0

29.04

13

24

-3.59

2.34 mM

-0.33

5×102

0

31.84

14

25

-3.55

2.48 mM

-0.32

4.1×102

0

23.65

15

47

-3.34

3.06 mM

-0.31

2.3×102

0

29.66

16

50

-3.19

4.57 mM

-0.29

2.8×102

0

27.47

17

19

-3.11

5.25 mM

-0.28

1.5×102

0

30.21

18

14

-2.88

7.74 mM

-0.26

1.7×102

0

26.56

19

15

-2.81

8.75 mM

-0.26

102

0

24.7

20

4

-2

12.38 mM

-0.24

2.6×10

0

33.32

18

Figure legends Fig. 1. Chemical structure of pregabalin. Fig. 2. Pregabalin (S-3-(aminoethyl)-5-methylhexanoic acid (CAS 148553-50-8) and the structurally related, endogenous molecules GABA and L-leucine. Fig. 3. Absorption spectra of DNA (5×10-5 M) in the absence and presence of increasing amounts of pregabalin (ri = [DNA] / [drug] = 0.142-4.4). Fig. 4. Fluorescence spectra of the competition between pregabalin and Hoechst. Cdrug = 0.614, 1.05, 1.4, 2.02, 2.4, 2.8, 3.3 and 3.7 × 10 -4mol L-1 for curves 2-9, C Hoechst = 5.0 × 10 -6 mol L-1, and CDNA = 2.5 ×10 -4 mol L-1 at 310 K. Fig. 5. Effect of increasing concentration of drug on the relative viscosity of CT-DNA at 25 ºC. Fig. 6. 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, 0.1, 0.2). Fig. 7. Molecular docked model of pregabalin showing groove binding with DNA

19

Fig. 1. Chemical structure of pregabalin

Fig. 2. Pregabalin (S-3-(aminoethyl)-5-methylhexanoic acid (CAS 148553-50-8) and the structurally related, endogenous molecules GABA and L-Leucine.

20

Fig. 3. Absorption spectra of DNA (5×10-5 M) in the absence and presence of increasing amounts of pregabalin: (ri = [DNA] / [drug] = 0.142 _ 4.4)

Fig.4 : Fluorescence spectra of the competition between pregabalin and Hoechst. CHoechst = 5.0 × 10-6 mol L_1, and CDNA = 2.5 ×10-4 mol L-1at 310 K.

21

Fig. 5. Effect of increasing concentration of drug on the relative viscosity of CT-DNA at 25 ºC

Fig. 6. CD spectra of DNA (5×10-5 M) in 10 mM Tris-HCl buffer, in the presence of increasing amounts of drug (ri = [drug]/[DNA] =0.0, 0.1, 0.2) 22

Fig. 7 : Molecular docked model of pregabalin showing groove binding with DNA

23

Research Highlights: •

Binding of pregabalin to DNA was studied using multispectroscopic methods and moleculr docking

•

Hydrogen bond and van der Waals play main roles in the binding of pregabalin to CTDNA.

•

The docking results revealed that groove mechanism is followed by pregabalin to bind with DNA.

•

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

24

25