Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study

Accepted Manuscript Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular mode...

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Accepted Manuscript Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study Yusra Rahman, Shumaila Afrin, Mohammed Amir Husain, Tarique Sarwar, Abad Ali, Shamsuzzaman, Mohammad Tabish PII:

S0003-9861(17)30136-4

DOI:

10.1016/j.abb.2017.05.014

Reference:

YABBI 7481

To appear in:

Archives of Biochemistry and Biophysics

Received Date: 25 February 2017 Revised Date:

15 May 2017

Accepted Date: 25 May 2017

Please cite this article as: Y. Rahman, S. Afrin, M.A. Husain, T. Sarwar, A. Ali, Shamsuzzaman, M. Tabish, Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug, with calf thymus DNA: An in vitro and molecular modelling study, Archives of Biochemistry and Biophysics (2017), doi: 10.1016/j.abb.2017.05.014. 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.

ACCEPTED MANUSCRIPT 1

Unravelling the interaction of pirenzepine, a gastrointestinal disorder drug,

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with Calf thymus DNA: An in vitro and molecular modelling study.

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Yusra Rahmana, Shumaila Afrina, Mohammed Amir Husaina,c, Tarique

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Sarwara,c, Abad Alib, Shamsuzzamanb and Mohammad Tabish*a

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a

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202002, India

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b

Department of Biochemistry, Faculty of Life Sciences, A.M. University, Aligarh, U.P.

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Aligarh 202002, India.

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c

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Steroid Research Laboratory, Department of Chemistry, Aligarh Muslim University,

Present address: Department of Biosciences, Jamia Millia Islamia, New Delhi-110025

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*Corresponding author:

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Department of Biochemistry

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Faculty of Life Sciences

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A.M. University, Aligarh

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U.P. 202002, India

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Email: [email protected]; Tel: +91-9634780818

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No. of figures: 10

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No. of tables: 3

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Total pages: 24

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ACCEPTED MANUSCRIPT Abstract

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Pirenzepine is an anti-ulcer agent which belongs to the anti-cholinergic group of

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gastrointestinal disorder drugs and functions as an M1 receptor selective antagonist. Drug-

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DNA interaction studies are of great significance as it helps in the development of new

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therapeutic drugs. It provides a deeper understanding into the mechanism through which

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therapeutic drugs control gene expression. Interaction of pirenzepine with calf-thymus DNA

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(Ct-DNA) was determined via a series of biophysical techniques. UV-visible absorption and

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fluorescence spectroscopy confirmed the formation of pirenzepine-Ct-DNA complex. The

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values of binding constant from various experiments were calculated to be in the order of 103

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M-1 which is consistent with the groove binding mode. Various spectrofluorimetric

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experiments like competitive displacement of well known dyes with drug, iodide quenching

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experiments and the effect of Ct-DNA denaturation in presence of drug confirmed the

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binding of pirenzepine to the groove of Ct-DNA. The binding mode was further established

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by viscometric, circular dichroic and molecular modelling studies. Thermodynamic

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parameters obtained from isothermal titration calorimetric studies suggest that the interaction

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of pirenzepine with Ct-DNA is enthalpically driven. The value of T∆S and ∆H calculated

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from calorimetric studies were found to be 4.3 Kcal mol-1 and -2.54 Kcal mol-1 respectively,

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indicating that pirenzepine-Ct-DNA complex is mainly stabilized by hydrophobic interaction

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and hydrogen bonding. The binding energy calculated was -7.5 Kcal mol-1 from modelling

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studies which was approximately similar to that obtained by isothermal titration calorimetric

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studies. Moreover, the role of electrostatic interaction in the binding of pirenzepine to Ct-

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DNA cannot be precluded.

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Keywords: Pirenzepine; calf- thymus DNA; groove binding; isothermal titration calorimetry;

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molecular modelling.

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ACCEPTED MANUSCRIPT 1 Introduction

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Deoxyribonucleic acid, the carrier of genetic information, is involved in indispensable life

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processes like gene transcription, gene expression, recombination and cell death. It is one of

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the major intracellular targets of small molecule, primarily due to the accessibility of its

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genome sequence, its well analysed three dimensional structure and the certainty of its

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functional and chemical groups. The interaction of small molecules has gained attention in

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recent years and has become one of the major research subjects.1 Small molecules alter the

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functioning of DNA by targeting its specific sequences, thus regulating the protein synthesis,

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expression of genes and growth of cells. In this way, small molecules can modify, inhibit or

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activate the functioning of DNA and thus act as therapeutics for the treatment and prevention

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of diseases.2 Indeed, many of them are approved clinically as therapeutic agents while some

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of them are in their advanced clinical trials.3 The importance of studying drug-DNA

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interaction is to gain insight into its mode of action at the molecular level, the origination of

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some diseases and is also helpful in the repositioning of drugs as anti-cancer agents.4

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Moreover, drug-DNA interaction studies also optimize the clinical efficacy of the existing

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drugs, thus advantageous in understanding the toxicological and pharmacological effect of

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the drug. This leads to the design and development of new effective drugs.5

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Small molecules can interact with DNA both covalently and non-covalently. Covalent

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interactions have more deleterious effects and are irreversible in nature whereas non covalent

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interactions are reversible.6 Non-covalent interactions include: (a) electrostatic binding which

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involves the interaction of negative phosphate groups of DNA with the positive end of small

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molecule (b) groove binding which involves hydrogen bonding and van der Waal’s

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interaction with nucleic acids (c) intercalative binding which occurs when small molecules

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intercalate within the stacked base pairs of DNA. Binding between small molecules and DNA

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can also involve intercalative as well as groove binding mode and this property can be related

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to their clinical efficacy and mode of action.7

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Pirenzepine,11-[(4methylpiperazin1yl)acetyl]5,11dihydro6Hpyrido[2,3b][1,4]benzodiazepin-

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6-one], is an anti-ulcerative M1 muscarinic antagonist, belonging to the anti-cholinergic

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class of gastrointestinal drugs. Being M1 receptor antagonist, it binds to the muscarinic

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acetylcholine receptor and mediates diverse range of cellular responses, suppressing acid

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output. It is used to treat gastric and peptic ulcers clinically.8 Also, it has been shown to

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suppress the elevating levels of serum gastrin by acting on gastrin cells (G cells) either

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directly or indirectly.9 Pirenzepine also promotes the healing of duodenal ulcers and due to

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ACCEPTED MANUSCRIPT its cytoprotective nature, it prevents the recurrence of duodenal ulcers. It also enhances the

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effect of other antiulcer agents like ranitidine and cimetidine. The M1 muscarinic effect of

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pirenzepine is considered to be responsible for the vago-mimetic neuro-humoral regulation,

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useful in the treatment of chronic heart failure and hypertension.10 Other therapeutic uses of

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pirenzepine include co-medication with anti-psychotic drugs.11 It is also administered

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together with drugs like olanzapine or clozapine to suppress side effects in cancer or

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schizophrenia treatments.12 It has also been found to be effective in reducing the progression

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of myopia, especially in children.13

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The present work was carried out to delineate the interaction mechanism of pirenzepine with

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Ct-DNA. Despite pirenzepine’s various therapeutic properties, its binding studies with DNA

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have not been investigated. The mode of binding of pirenzepine with Ct-DNA was

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ascertained through various biophysical and molecular modelling studies. UV-visible

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absorbance and steady-state fluorescence spectroscopy was employed to assess the formation

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and stability of pirenzepine-Ct-DNA complex. Various fluorimetric studies like KI quenching

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experiments, competitive displacement assays and effect of denaturation of DNA was

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performed which indicated the binding mode of pirenzepine to Ct-DNA. Furthermore,

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hydrodynamic technique like viscometric studies, DNA melting studies and circular dichroic

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studies validated the mode of binding of pirenzepine. Thermodynamic parameters were

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determined from the isothermal titration calorimetric studies which provide additional

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support to the binding mechanism. Molecular modelling studies were also performed to gain

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deeper knowledge into the binding mode and also the relative binding energy of pirenzepine-

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Ct-DNA complex.

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2 Experimental

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2.1 Materials

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Pirenzepine dihydrochloride, calf thymus DNA (Ct-DNA), 4', 6-diamidino-2-phenylindole

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(DAPI) and Hoechst-33258 were purchased from Sigma Aldrich, USA. Ethidium bromide

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was purchased from Himedia, India. All other chemicals and solvents used were of reagent

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grade. All experiments were performed in 10 mM Tris-HCl buffer (pH 7.4).

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2.2 Sample preparation

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The stock solution of pirenzepine (10 mM) was prepared in distilled water and the working

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solutions were prepared as required. Ct-DNA was dissolved in 10 mM Tris-HCl (pH 7.4) and

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stored at 4 °C for 24 hrs with periodic stirring to ensure the formation of a homogenous

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solution. Absorbance ratio A260nm/A280nm was found to be >1.8 implying that solution of Ct-

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ACCEPTED MANUSCRIPT DNA was free from protein. The final concentration of DNA solution was measured using

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extinction coefficient of 6600 M−1 cm−1 of a single nucleotide at 260 nm.14

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2.3 UV-Vis spectroscopic titrations

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The UV spectra were measured on Shimadzu dual beam UV-visible spectrophotometer

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(Japan) using quartz cuvettes with path length of 1 x 1 cm. The UV spectra of pirenzepine

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and the pirenzepine-Ct-DNA complex were scanned in the wavelength range of 230-300 nm.

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The measurements were carried out in the presence of a fixed concentration of pirenzepine

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(50 µM) by titrating with increasing concentrations of Ct- DNA (0-56 µM). 10 mM Tris-HCl

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was used to make up the total volume of 3 mL and is used for baseline correction as well.

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2.4 Fluorescence spectroscopic studies

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Fluorescence measurements were carried out on a Shimadzu spectrofluorometer-5000

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(Japan) equipped with a xenon flash lamp using 1.0 cm quartz cells. The emission spectra of

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pirenzepine were recorded from 400 to 550 nm upon excitation at 281 nm with the excitation

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and emission slit widths set at 5 nm each. The concentration of pirenzepine was kept fixed at

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50 µM with varying the concentration of Ct-DNA from 0-32 µM in a total volume of 3 mL.

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2.5 Potassium iodide quenching experiments

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Potassium iodide quenching experiments of pirenzepine were performed in the absence and

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presence of Ct-DNA. In the first set, pirenzepine alone was titrated with increasing

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concentrations of KI (0 to 52.8 µM). In another set, pirenzepine (50 µM) in presence of Ct-

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DNA (50 µM) were titrated with increasing concentrations of KI (0-52.8 µM). Excitation was

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performed at 281 nm and fluorescence emission was recorded from 400 to 550 nm. Stern-

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Volmer equation was applied to calculate the fluorescence quenching efficiency.

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2.6 Competitive displacement studies

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Ethidium bromide displacement assay was performed by using a solution containing 5 µM

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ethidium bromide with 50 µM Ct-DNA and was titrated with increasing concentrations of

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pirenzepine from 0 to 200 µM. The emission spectra were recorded in the range 520-700 nm

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upon excitation of the ethidium bromide-Ct-DNA complex at 475 nm.

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Similarly, Hoechst displacement assay was performed. The Hoechst-Ct-DNA complex

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containing 5 µM Hoechst-33258 and 50 µM Ct-DNA was excited at 343 nm and fluorescence

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emission spectra were recorded from 380-600 nm by titrating with increasing concentrations

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of pirenzepine from 0 to 200 µM.

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DAPI displacement assay was also performed similarly as ethidium bromide and Hoechst

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displacement assays. DAPI-Ct-DNA complex containing 5 µM DAPI and 50 µM Ct-DNA

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was titrated with increasing concentrations of pirenzepine from 0 to 200 µM. DAPI-Ct-DNA

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ACCEPTED MANUSCRIPT complex was excited at 358 nm and the fluorescence emission spectra were recorded from

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380-600 nm.

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2.7 Comparison of interaction of pirenzepine with ssDNA and dsDNA.

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Effect of denaturation of DNA was studied by heating the double stranded DNA on a boiling

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water bath for 30 min and then readily cooling on an ice bath for 10 min. Experiments were

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completed within 3 hrs so that single-stranded DNA is not converted into double stranded

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DNA. Pirenzepine (50 µM) was titrated with increasing concentrations of either ssDNA or

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dsDNA. Excitation was done at 281 nm and the emission spectra were recorded in the range

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of 400-550 nm.

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2.8 Effect of Ionic strength

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Effect of ionic strength was studied by varying the concentration of NaCl from 0 to 29.7 mM

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in the solution of total volume of 3 mL containing fixed concentration of pirenzepine and Ct-

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DNA (50 µM each). Emission spectra were recorded between 400-550 nm and excitation

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wavelength was fixed at 281 nm.

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2.9 Viscosity measurements

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Viscosity measurements were performed by using an Ubbelohde viscometer suspended

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vertically in a thermostat at 25 °C (accuracy ± 0.1 oC). The flow time was determined using a

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digital stopwatch and each sample was tested three times to get an average calculated time.

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Here, DNA concentration was kept constant (50 µM) and the viscosity was measured in the

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absence and presence of increasing concentrations of pirenzepine. Data was represented as

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(/0)1/3 versus ratio of pirenzipine/DNA, where  and 0 are the viscosities of DNA in the

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presence and absence of pirenzepine, respectively.15

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2.10 Circular dichroism (CD) studies

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CD spectra were recorded by using Applied Photophysics CD spectrophotometer (model

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CIRASCAN, UK) equipped with a Peltier temperature controller to maintain the sample’s

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temperature constant at 25 oC. The molar ratio of DNA concentration to pirenzepine

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concentration taken were 1:0, 1:1. 1:2. All the CD spectra were determined in the wavelength

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range of 220-310 nm with a scanning speed of 200 nm/min and a spectral band width of 10

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nm. The background spectrum of buffer solution (10 mM Tris-HCl, pH 7.4) was subtracted

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from the spectra of DNA and pirenzepine-Ct-DNA complex. Each spectrum was the average

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of three scans.

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2.11 DNA melting studies

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DNA melting studies were carried out by monitoring the absorbance of different

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concentrations of Ct-DNA:pirenzepine (0,1:1,1:2) at 260 nm at various temperatures ranging

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ACCEPTED MANUSCRIPT from 25-90 oC using a spectrophotometer attached with a thermocouple. The total volume of

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the sample was made to 3mL using using Tris-HCl (10 mM, pH 7.4). The absorbance was

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plotted as a function of temperature and Tm was obtained as a transition midpoint from the

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melting curve.

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2.12 Isothermal titration calorimetry

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The thermodynamics of the pirenzepine-Ct-DNA interaction was studied at 303 K using VP-

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ITC microcalorimeter (Microcal Inc., Northampton,MA). Prior to loading, samples were

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degassed in a thermovac for 10 min to eliminate air bubbles. Pirenzepine (2.25 mM) was

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successively injected with volume of 10 µL into the sample cell containing 0.05 mM Ct-DNA

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solution. 29 injections were made with an initial delay of 60 sec and each was made over 10

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sec with a spacing of 180 sec between consecutive injections. The reference power and the

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stirring speed were fixed at 16 µcals-1 and 307 rpm respectively. The calorimetric data were

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analysed using Microcal origin 7.0 software which is provided with the instrument. The

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thermodynamic parameters were calculated using the formula

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∆G= RT ln Kb =∆H-T∆S

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Where T is the absolute temperature at 303 K and R is the gas constant which has a value of

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1.987 cal/Kmol.

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2.13 Molecular docking studies

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Molecular docking studies were carried out using Autodock 4.0 to study the interaction of

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pirenzepine with Ct-DNA. The Lamarckian genetic algorithm used for calculations were

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employed in Autodock 4.0 which was proven to be more reliable, sensitive and effective.16

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The structure of B-DNA dodecamer d(CGCGAATTCGCG)2 with (PDB ID: 1BNA) was

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downloaded from protein data bank (http://www.rcsb.org./pdb). The three-dimensional

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structure of pirenzepine was obtained from (https://pubchem.ncbi.nlm.nih.gov) in .sdf format

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which was converted to .pdb format by Avogadro’s 1.01.17 All water molecules were

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removed before performing docking and Kollmann charges were introduced along with the

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polar hydrogen atoms. The size of the grid was set to 54, 56 and 54 along the x-axis, y-axis

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and z-axis respectively, with spacing of 0.419 Å having centre of grid at x=14.421, y=

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17.959, z= -9.201. Out of different conformers docked, conformer with the minimum energy

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was taken into account. Docked conformation analysis was done using UCSF Chimera 1.01,

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Accelrys Discovery Studio 4.5 and PyMOL.

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ACCEPTED MANUSCRIPT 3 Result and discussion

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3.1 Determination of interaction of pirenzepine with DNA

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3.1.1 UV–Visible spectroscopy

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UV-visible absorption spectroscopy is a simple and efficient method to detect the formation

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of complex between small molecules and DNA. The complex formation is accompanied by

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changes in the position of peak and absorption spectra of the molecule.18 The absorption

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spectra of pirenzepine showed maxima at 281 nm (Fig 1a). Addition of increasing

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concentrations of Ct-DNA to pirenzepine resulted in hyperchromism with a noticeable blue

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shift19 of 9 nm (281nm-272 nm) in the peak position of pirenzepine. The hyperchromism

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observed is due to unwinding of double helix of DNA as a result of its interaction with

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pirenzepine and denotes the formation of complex between Ct-DNA and pirenzepine. Non-

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covalent interactions involving mainly electrostatic and groove binding (interactions outside

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the DNA helix) are generally marked by hyperchromism. Small molecules intercalating

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within the base pairs of DNA have been found to show hypochromism accompanied by red

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shift.3 It can therefore be deduced that there is a possibility of pirenzepine interacting with Ct-

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DNA by groove binding and electrostatic binding mechanism.

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Another experiment was carried out where Ct-DNA (50 µM) was kept constant and titrated

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with varying concentrations of pirenzepine (20 µM to 60 µM). Hyperchromicity with

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insignificant blue shift was observed on addition of increasing concentration of drug (Fig 1b)

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indicating Ct-DNA-pirenzepine complex formation.

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The association constant was determined using the Benesi-Hildebrand equation20 given

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below:

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=



  

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[ ]



(1)

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Where, A0 is the absorbance of pirenzepine in the absence of Ct-DNA and A is the

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absorbance of pirenzepine in the presence of different concentrations of Ct-DNA.  and

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 are the absorption co-efficient of pirenzepine and the pirenzepine-Ct-DNA complex,

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respectively. The plot of A0/A-A0 termed as double reciprocal plot is linear (Fig 1c). The

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value of association constant was estimated from the ratio of the intercept to slope and found

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to have a value of 1.57 x 103 M-1 signifying interaction between pirenzepine and Ct-DNA.

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ACCEPTED MANUSCRIPT 3.1.2 Steady State Fluorescence

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Fluorescence emission spectroscopy offers a sensitive and selective approach to study the

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interactions between small molecules and DNA.18 A series of fluorescence based studies have

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been employed to determine the strength as well as the binding mode of pirenzepine with Ct-

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DNA.

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Steady state fluorescence was performed to understand the formation of pirenzepine-Ct-DNA

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complex. Due to the poor intrinsic fluorescence of DNA, pirenzepine has been used as a

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fluorophore to study its interaction with Ct-DNA. The emission wavelength of pirenzepine

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revealed a peak at 445 nm, when excited at its excitation maxima at 281 nm (Fig 2a). The

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fluorescence spectra of pirenzepine in the presence of Ct-DNA was marked by an increase in

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the fluorescence intensity as compared to free pirenzepine, confirming the formation of

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complex between Ct-DNA and pirenzepine. Moreover, the binding of pirenzepine to Ct-DNA

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undergoes a red shift, suggestive of change to a more polar environment.21 Intercalative

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binding, involving the constraint in the rotational motion of the base pairs of DNA, favours

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the radiation less deactivation of the excited molecules resulting in the subsequent decrease in

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their fluorescence intensity. While in case of molecules binding to the grooves of DNA, the

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deactivation by fluorescence emission is favoured which results in the increase in

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fluorescence intensity.22 Enhancement in the fluorescence intensity of pirenzepine on

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addition of Ct-DNA was observed, indicating the groove binding mode as the possible mode

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of interaction.

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The enhancement constant [E] which is analogous to the quenching constant [Q] of the

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quenching process, was calculated from the Stern-Volmer equation21 as given below:

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F0/ F = 1−KD [E] or F0/ F = 1−KS [E]

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(2)

Where KD is the dynamic enhancement constant and KS is the static enhancement constant. F0

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and F signifies the fluorescence intensities in the absence and presence of enhancer [E] i.e.

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Ct-DNA, respectively. The value of KD or KS was determined from the slope of the plot of

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F0/F versus concentration of enhancer (Fig 2b). It was calculated to be 2.3 x 104 M-1 which is

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consistent with the binding constant of groove binders.23 The linear Stern-Volmer plot

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obtained from the fluorescence study suggests the occurrence of only one type of binding.24

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In order to differentiate between the static and dynamic enhancement, the bimolecular

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enhancement constant, KQ (analogous to the bimolecular quenching constant) is calculated

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from the equation given below where τ0 is the fluorophore’s lifetime in the absence of

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enhancer.

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ACCEPTED MANUSCRIPT KD (KS) = KQ .τ0

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(3)

Since, the fluorescence lifetimes are typically near 10-8 s, the bimolecular enhancement

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constant (KQ) was calculated from the above equation and found to be 2.3 x 1012 M-1 s-1. The

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value obtained is larger than the largest possible value for dynamic enhancement which is 1 ×

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1010 M−1 s−1. Thus, the fluorescence enhancement is not initiated by the dynamic process, but

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due to the static process with ground state complex formation.25

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The binding constant (K) and the number of binding sites (n) were determined from the inset

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(fig 2b), using the equation.26



= log K + n log [DNA]

(4)

In the case of enhanced fluorescence intensity, F0 < F, this equation becomes Log

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= log K + n log [DNA]

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Where, F and F0 are the fluorescence intensities of the fluorophore in the presence and

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absence of enhancer i.e. Ct-DNA, respectively. The values of K and n were calculated to be

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3.1 x 103 M-1 and 0.812, respectively from the plot of log (∆F/F) versus log [DNA].

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3.2 Determination of the mode of binding of pirenzepine with DNA

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3.2.1 Iodide quenching studies

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Iodide ion is an anionic quencher which adequately quenches the fluorescence of small

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molecules in an aqueous environment. Fluorescence quenching experiment using iodide ion

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as a quencher was selected to determine the binding mode of pirenzepine with DNA. Small

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molecules bound to the groove are more exposed to the aqueous environment surrounding the

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helix and are not well protected; therefore they are readily quenched by the iodide ions even

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in the presence of DNA. In intercalative mode of binding, small molecules are protected from

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quenching by iodide ions due to their stacking between the base pairs and repulsion between

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negatively charged iodide ion and phosphate backbone of DNA.22 It was observed that

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pirenzepine was quenched by iodide ion in the absence and presence of Ct-DNA (Fig 3). The

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quenching constant Ksv, was calculated from the Stern-Volmer plot and equation as

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described below:

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F0/F = 1+ Ksv [Q]

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ACCEPTED MANUSCRIPT Here, F0 and F are the fluorescence intensities in the absence and presence of the quencher.

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[Q] is the concentration of the quencher and Ksv is the quenching constant. The Ksv values of

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pirenzepine with iodide ions (1.5 x 103 M-1) decreased slightly in the presence of Ct-DNA

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(1.41 x 103 M-1). This little decrease (Table 1) in the quenching of pirenzepine by iodide ions

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can be ascribed to the fact that the pirenzepine binding to the groove of DNA is partially

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protected and consequently, there is a little decrease in the Ksv value compared to

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pirenzepine alone. In case of intercalation, the magnitude of Ksv of free drug would be much

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higher than that of drug bound to DNA due to its intercalation within base-pairs.27 Thus, it

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suggests that pirenzepine does not intercalate rather it binds to the grooves of Ct-DNA.

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3.2.2 Competitive displacement assay

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Fluorescence based competitive dye displacement assay using dyes with established binding

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mode were employed to further verify the binding mode of pirenzepine to Ct-DNA. If the

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binding of small molecule to DNA displaces the dye bound to the DNA, it can be deduced

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that it interacts with the DNA in the similar manner as the displaced dye. The addition of

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small molecule to the DNA-dye system provides valuable information regarding the binding

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mode as it changes the fluorescence behaviour of the system. Ethidium bromide (3, 8-

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Diamino-5-ethyl-6-phenylphenanthridinium bromide) is a phenathridine fluorescent dye

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which interacts with DNA by intercalating within its base pairs.28 Ethidium bromide does not

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considerably manifest any fluorescence in the buffer solution due to its quenching by solvent

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molecules. However, in the presence of DNA, fluorescence intensity of ethidium bromide is

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increased due to intercalation of its planar phenanthridinium ring between base pairs of the

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double helix. To determine the binding mode of pirenzepine, displacement assay with

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ethidium bromide was performed. On continuous addition of pirenzepine to the ethidium

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bromide-Ct-DNA complex, there was no significant decrease in the fluorescence intensity

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(Fig 4a) which suggests that pirenzepine was not binding in the same manner as ethidium

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bromide i.e. it does not bind through the intercalative mode of binding.

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To establish the groove binding mode of pirenzepine, displacement assay with Hoechst were

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carried

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benzimidazo-2-yl]-benzimidazole derivative, binds to the minor groove of DNA.18 Hoechst

347

shows weak fluorescence in buffer due to its quenching by the solvent molecules. However,

348

its fluorescence intensity is significantly increased when it binds to the minor groove of

349

DNA. This occurs due to its planar nature and its protection from collisional quenching.29

350

Successive addition of pirenzepine to the Hoechst-Ct-DNA system, results in marked

351

decrease in the fluorescence intensity (Fig 4b). This quenching can be explained on the basis

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out.

Hoechst

33258

[2-(4-hydroxyphenyl)-5-[5-(4-methylpipera-zine-1-yl)

ACCEPTED MANUSCRIPT of pirenzepine competing with Hoechst-33258, thus replacing it from the minor groove and

353

binding in a similar manner.

354

To further confirm the groove binding of pirenzepine, displacement assay with DAPI have

355

been carried out. DAPI (4', 6-diamidino-2-phenylindole) is a well-known groove binder

356

which preferably binds to the minor groove of DNA.30 When DAPI binds to the minor groove

357

of DNA, its fluorescence intensity is increased as compared to DAPI alone. On addition of

358

increasing concentrations of pirenzepine to the DAPI-Ct-DNA system, quenching in the

359

fluorescence intensity of the system was observed (Fig 4c). Thus, pirenzepine displaces DAPI

360

bound to the minor grooves of DNA. This quenching of fluorescence intensity of pirenzepine

361

on both Hoechst-Ct-DNA system and DAPI-Ct-DNA system confirmed the groove binding

362

of pirenzepine to Ct-DNA.

363

The quenching of ethidium bromide, DAPI and Hoechst bound to Ct-DNA by pirenzepine

364

was also estimated in terms of Ksv from the Stern-Volmer equation3 (Fig 4d). From table 2, it

365

is evident that the Ksv value in case of DAPI and Hoechst is much higher as compared to

366

ethidium bromide. Thus, it can be inferred from these experiments that pirenzepine is binding

367

in the groove of Ct-DNA.

368

3.2.3 Effect of denaturation of DNA

369

In this experiment, the effect of binding of single stranded DNA on the fluorescence of

370

pirenzepine was explored. Single stranded DNA was obtained by heating the double stranded

371

DNA in a boiling water bath for 30 min and then readily cooling in on ice bath for 10 min.

372

Single stranded DNA obtained from this was used within 3 hrs before it gets renatured to

373

double stranded DNA. The fluorescence intensity of pirenzepine was enhanced in presence of

374

single stranded DNA and this enhancement in fluorescence intensity was slightly greater than

375

the enhancement in fluorescence intensity observed in presence of double stranded DNA

376

under similar conditions, suggesting that pirenzepine interacts with the bases of DNA through

377

hydrogen bonds in minor groove. Fig 5 shows the relative fluorescence intensities of

378

pirenzepine with increasing concentrations of single stranded and double stranded DNA. This

379

observation can be attributed to the fact that the enhancement of fluorescence intensity by

380

single stranded DNA cannot be accomplished in case of intercalators owing to the absence of

381

base pairs in single stranded DNA. In case of groove binders, although the major grooves and

382

minor grooves are destroyed by denaturation of double stranded DNA but the single stranded

383

DNA has a line-and-knot conformation and the knot has concave surfaces which can form

384

hydrogen bonds with small molecules. So, there is an enhancement in fluorescence still in the

385

presence of single stranded DNA.31 Earlier, in the competitive displacement assay, we have

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ACCEPTED MANUSCRIPT observed that pirenzepine is capable of displacing the known minor groove binders (DAPI,

387

Hoechst) from Ct-DNA, hence it interacts with the base-pairs in the minor grove through

388

hydrogen bonds and hydrophobic interactions and not interacting with structural component

389

in dsDNA. In case of electrostatic binding which takes place outside the DNA helix and the

390

change in the fluorescence intensities in presence of single and double stranded DNA should

391

be the same. Thus, the enhancement in fluorescence intensity of pirenzepine in presence of

392

single stranded DNA provides evidence that pirenzepine binds to grooves of Ct-DNA.

393

3.2.4 Viscosity measurements

394

Although spectroscopic methods are important to study the interaction of DNA with small

395

molecules, viscosity measurements provide a more concrete evidence to validate the mode of

396

binding.32 To further elucidate the binding mode of pirenzepine with Ct-DNA, viscometric

397

study was performed which detects the change in length of DNA upon addition of ligand. In

398

case of intercalative binding mode, total viscosity of DNA solution is increased due to

399

accommodation of the small molecule between adjacent base pairs of the double helix,

400

resulting in increase in the overall length of DNA. Groove binding molecule or molecule

401

interacting through electrostatic mode cause little or no change in the viscosity of DNA

402

solution.33 To determine any changes in the viscosity, a plot of

403

[pirenzepine/DNA] was obtained (Fig 6a). With the continuous addition of pirenzepine to the

404

Ct-DNA solution, no significant increase in the overall length of Ct-DNA was observed

405

which indicates that the pirenzepine is interacting via external mode i.e. it binds in the groove

406

of Ct-DNA.

407

3.2.5 Circular Dichroism

408

Although DNA exists in a number of conformations like A-form, Z-form, B-form,34

409

however, the B-form of DNA is regarded as the prevalent physiological form. The

410

determination of variation in conformation of DNA upon interaction of small molecules is

411

widely studied by circular dichroic studies. The CD spectra of the B-form of DNA exhibit a

412

positive peak at 277 nm which corresponds to base pair stacking and a negative peak at 245

413

nm corresponding to its right-handed helicity.35 The intensity of both the bands are increased

414

with intercalators due to stabilization of the double helix of B-DNA whereas little or no

415

perturbation in the structural conformation of DNA is observed with groove binding and/or

416

electrostatic interacting molecules.36 To further confirm the mode of binding of pirenzepine,

417

CD spectra of Ct-DNA is recorded with increasing concentration (0-100 µM) of pirenzepine

418

(Fig 6b).There was no obvious changes in the CD spectra confirming the groove binding of

419

pirenzepine with Ct-DNA.

(/0)1/3 versus

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ACCEPTED MANUSCRIPT 3.2.6. DNA melting studies

421

The stability of double helix of DNA is attributed to hydrogen bonding and base stacking

422

interactions. On weakening of the forces involved in binding, the double helix is denatured.

423

The temperature at which half of the double helical strand of DNA is denatured to single

424

stranded DNA is referred to as the melting temperature (Tm) and relates to the stability of

425

DNA double helix. Small molecules interacting with DNA are known to affect its melting

426

temperature. Molecules which intercalate within the base-pairs of DNA stabilise the double

427

helix and increases the Tm by about 5-8 oC whereas molecules binding through electrostatic

428

interaction or in the groove of DNA causes little or no substantial increase in Tm.3 The Tm

429

value for Ct-DNA in the absence and presence of different concentrations of pirenzepine was

430

determined from the plot of A/A25oC versus temperature by monitoring the absorbance at 260

431

nm. Where A25 is the absorbance measured at 25 oC and A is the absorbance measured at

432

increasing temperatures ranging from 25oC to 90oC. The Tm for each transition was

433

determined as the transition midpoint of the melting curve. The value of Tm for Ct-DNA

434

alone was found out to be 77.75 ± 1 oC, whereas in presence of pirenzepine (1:1), Tm was

435

calculated to be 79.7 ± 1 oC and in the presence of pirenzepine (1:2), the Tm was 79.15 ± 1 oC

436

(Fig 7). The change in Tm value on interaction of different ratios of DNA and pirenzepine is

437

not significant enough thus, supporting the non-intercalative binding. Little increase in Tm of

438

Ct-DNA (approximately 2 oC) as a result of pirenzepine addition is probably due to the

439

binding of pirenzepine in the groove of Ct- DNA and changing its conformation. 37

440

3.2.7 Isothermal titration calorimetry

441

Isothermal titration calorimetry (ITC) is an effective method for the characterization of

442

thermodynamic parameters of non-covalent interactions involved between small molecules

443

and biological macromolecules. It also provides deeper insight into the energetics involved in

444

the interaction between different molecules.38 Hence, ITC was performed to analyse the

445

thermodynamic parameters in the formation of pirenzepine-Ct-DNA complex. The ITC

446

profile of the binding of pirenzepine with Ct-DNA is illustrated (Fig 8). The upper panel

447

shows the raw ITC profile of the pirenzepine injecting into the Ct-DNA at 30 oC. Each peak

448

in the isotherm corresponds to a single injection of pirenzepine into the Ct-DNA solution.

449

The lower panel represents the heat released per injection plotted as a function of the molar

450

ratio of pirenzepine to Ct-DNA. The isotherm was fitted to one site model in order to obtain

451

the best fit. The thermodynamic parameters obtained from ITC are represented in table 3. The

452

value of binding constant 1.0 x 103 M-1 was obtained which is comparable to that obtained

453

from spectroscopic and spectrofluorimetric studies.

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ACCEPTED MANUSCRIPT The large negative enthalpy change (∆H) accompanied by negative free energy change, ∆G

455

with a value of -6.7 Kcal mol-1 indicates that the reaction is enthalpically driven. It is

456

established in the earlier studies that groove binding interactions are an enthalpically driven

457

process whereas intercalative interactions are entropically driven.39 Further, the number of

458

binding sites of pirenzepine on Ct-DNA was found out to be near unity. The binding was

459

characterised by negative enthalpy change which indicates that the interaction between

460

pirenzepine and Ct-DNA is exothermic in nature and involves electrostatic interactions.

461

The positive value of T∆S and negative value of ∆H implies that the hydrophobic interactions

462

41

463

3.2. 8 Role of ionic strength

464

The role of ionic strength was studied to determine the involvement of electrostatic

465

interaction between pirenzepine and Ct-DNA. It is known that the intercalative and groove

466

binding modes are related with the double helix of DNA while electrostatic interaction takes

467

place out of the helix. NaCl is used to study the participation of electrostatic interactions

468

involved in the binding of pirenzepine to Ct-DNA. In presence of NaCl, electrostatic

469

repulsion between the negatively charged phosphate backbone of DNA is reduced. DNA

470

chains are thus constricted resulting in the protection of electrostatic interactions. Molecules

471

bound in the groove are exposed more to the external environment and can easily be released

472

from the helix on increasing the ionic strength as compared to the intercalators.

473

the participation of ionic strength becomes more relevant when there is no observable effect

474

on the fluorescence intensity of drug alone on addition of NaCl. The effect of increasing

475

concentrations of NaCl was observed on the fluorescence spectra of pirenzepine-Ct-DNA

476

complex. On addition of increasing concentration of NaCl to the pirenzepine-Ct-DNA

477

complex, an increase in fluorescence intensity was observed (Fig 9a). This is due to the

478

weakening of electrostatic interaction between pirenzepine and DNA and the release of

479

groove bound pirenzepine from the helix of DNA. These observations clearly indicate that

480

the binding of pirenzepine to Ct-DNA is predominantly controlled by electrostatic

481

interactions and are consistent with groove binding rather than intercalative mode of

482

interaction.

483

In order to analyse the participation of electrostatic forces, salt-dependency of pirenzepine

484

binding to Ct-DNA was studied at three different salt concentrations [Na+] at 10 mM, 20 mM

485

and 50 mM and the association constants were evaluated from Benesi-Hildebrand’s plot.43

486

The values of association constants corresponding to each salt concentrations i.e. at 10 mM,

487

20

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40

27

Studying

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and hydrogen bonding 42 are involved in the formation of pirenzepine-Ct-DNA complex.

mM

and

50

mM

obtained

were

3500

M-1,

2582

M-1

and

2053

M-1

ACCEPTED MANUSCRIPT 488

respectively. The following relationship was used to analyse the role of electrostatic forces in

489

binding process: log Ka/log [Na+] = -Z

490

(7)

Z is the apparent charge on the ligand and  is the fraction of [Na+] bound to phosphate

492

backbone of DNA. Straight line was obtained from the plot of log Ka and log [Na+] and from

493

the slope (Z), pirezepine released from DNA was calculated to be 0.32 (Fig 9b). As the

494

concentration of salt increases, there was a slight decrease with less variation in the value of

495

association constants. The results showed that binding of pirenzepine is accompanied by low

496

counter-ion release which is usually seen in case of molecules with neutral charge binding

497

with DNA molecule. 44

498

3.2.9. Molecular docking

499

Molecular docking studies are employed further to gain information in the binding mode of

500

pirenzepine to Ct-DNA as well as to substantiate the results of spectroscopic and calorimetric

501

experimental studies. Molecular docking is an effective tool to offer visual depiction for the

502

binding of small molecules to DNA and to understand the drug-DNA interactions for the

503

rational drug designing and discovery.31

504

Out of the 10 docking runs performed, a total number of seven best conformations was

505

obtained with their respective binding energies (Fig 10a). The conformer with the minimum

506

binding energy was picked from the total conformations obtained. As manifested (Fig 10b),

507

the most energetically favoured confirmation of pirenzepine docked in the minor groove of

508

Ct-DNA. Moreover, there are hydrogen interactions involving the –NH group of guanine

509

base pair (DG-10 of chain A) and O atom of DA-17 of chain B (Fig 10c). The lengths of the

510

hydrogen bonds are 2.8 and 2.6 Å, respectively (Fig 10d). Electrostatic interaction between

511

the negative charge phosphate backbone of Ct-DNA and pirenzepine was also depicted (Fig

512

10e). The free energy change of pirenzepine docked with DNA was found out to be-7.5 Kcal

513

mol-1. The slight discrepancy in the binding energy obtained by modelling and by ITC studies

514

could be due to the elimination of water molecules in molecular docking studies.

515

To further understand the mode of binding of pirenzepine to Ct-DNA, a competitive

516

molecular docking was performed using Hoechst docked to DNA. The benzyl group of

517

pirenzepine binds in the vicinity of the docked Hoechst (Fig 10f). This further corroborates

518

the groove binding of pirenzepine to Ct-DNA.

519 520

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4 Conclusion

522

In the present study, the binding mode of pirenzepine with Ct-DNA was established via

523

multi-spectroscopic, calorimetric and molecular modelling studies. Formation of complex

524

between pirenzepine and Ct-DNA was determined by UV-Visible absorbance and

525

fluorescence spectroscopy. The binding constant value was found to be approximately 103 M-

526

1

527

pirenzepine in presence of Hoechst and DAPI indicated the binding of pirenzepine to the

528

minor groove of Ct-DNA. The groove binding of pirenzepine was further validated by iodide

529

quenching, viscometric and circular dichroic studies. The binding process was found to be

530

spontaneous and the involvement of hydrophobic interaction and hydrogen bonding was

531

revealed by the thermodynamic parameters evaluated by ITC. The participation of

532

electrostatic interaction in the binding of pirenzepine to Ct-DNA was also determined by

533

studying the role of ionic strength. Molecular modelling studies further corroborated the

534

groove binding mode of pirenzepine.

535 536

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, providing clear evidence of groove binders. Quenching of the fluorescence intensity by

Notes

538

The authors declare that there is no conflict of interest in this work.

539

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Acknowledgement

541

We are thankful to UGC, New Delhi for MAF to YR, TS and MAH and to the CSIR, New

542

Delhi for providing JRF to SA. We are also thankful to the departments of Biochemistry and

543

Chemistry for providing the necessary facilities.

545 546 547 548 549 550 551

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Figure Legends

653

Fig. 1. (a) UV-visible absorption spectra of pirenzepine (50 µM) in absence and presence of

655

increasing concentrations of Ct-DNA (0-56 µM) in 10 mM Tris- HCl buffer (pH 7.4). (b)

656

UV-visible absorption spectra of DNA (50 µM) in absence and presence of increasing

657

concentrations of pirenzepine (0-60 µM) (c) A double reciprocal plot of binding of

658

pirenzepine to Ct-DNA. The chemical structure of pirenzepine dihydrochloride has been

659

represented in the inset. Data represents mean ± SD of three different experiments.

RI PT

654

Fig. 2.

661

(a) Steady-state fluorescence spectra of pirenzepine (50 µM) in absence and presence of

662

increasing concentrations of Ct-DNA (0-32 µM). (b) Stern-Volmer plot for the pirenzepine-

663

Ct-DNA interaction. Enhancement constant was found out to be 1.97 x 103 M-1. The inset

664

shows the modified Stern-Volmer plot of log [(F-F0)/F] versus log [DNA]. Data represents

665

mean ±SD of three experiments.

666

Fig. 3.

667

Stern-Volmer plot for the fluorescence quenching of pirenzepine by successive addition of KI

668

(0- 52.8 µM) in absence and presence of Ct-DNA (50 µM). Data represent mean ± SD of

669

three experiments.*p value <0.05 as compared to control.

670

Fig. 4.

671

(a) Fluorescence titration of ethidium bromide-Ct-DNA complex with increasing

672

concentration of pirenzepine. Ethidium bromide-Ct-DNA complex was excited at 476 nm and

673

emission spectra were recorded from 520-700 nm. (b) Fluorescence titration of Hoechst-Ct-

674

DNA complex with increasing concentration of pirenzepine. Hoechst-Ct-DNA complex was

675

excited at 343 nm and emission spectra were recorded from 380-600 nm. (c) Fluorescence

676

titration of DAPI-Ct-DNA complex with increasing concentration of pirenzepine. DAPI-Ct-

677

DNA complex was recorded from 380-600 nm. (d) Stern-Volmer plot for the fluorescence

678

intensity quenching of dyes-Ct-DNA complex on varying the concentration of pirenzepine

679

(0-220 µM). Data represent mean ± SD of three experiments.*p value < 0.05 as compared to

680

control.

681

Fig. 5.

682

Effect of single-stranded DNA and double stranded DNA on relative fluorescence intensities

683

of pirenzepine. F and F0 represents the fluorescence intensities with and without Ct-DNA,

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ACCEPTED MANUSCRIPT respectively. Data represent mean ±SD of three experiments.*p value < 0.05 as compared to

685

control.

686

Fig.6.

687

(a) Effect of increasing the concentration of pirenzepine on the viscosity of DNA. The

688

concentration of Ct-DNA (100 µM) was kept constant while varying the concentration of

689

pirenzepine. The values are mean of ± SD of three experiments.*p value < 0.05 as compared

690

to control. (b) Circular dichroic spectra of Ct-DNA (50 µM) in the absence and presence of

691

increasing concentrations of pirenzepine (1:0, 1:1. 1:2).

692

Fig.7.

693

DNA melting studies at different concentrations of DNA:pirenzepine at varying temperature

694

ranging from 25-90oC.

695

Fig.8.

696

Isothermal titration calorimetry profiles of pirenzepine interaction with Ct-DNA. (a) The

697

upper panel shows the raw ITC profiles of the injection of pirenzepine into the Ct-DNA

698

solution. The distribution of DNA binding enthalpy values normalized to total pirenzepine

699

per injection is shown in the inset. (b) The lower panel shows the integrated heat profile of

700

the calorimetric titration. The solid line represents the best fit data and the binding sites were

701

fit to a single site model.

702

Fig. 9.

703

(a)Role of ionic strength. Maximum intensity plot of pirenzepine-Ct-DNA complex with the

704

increasing concentration of NaCl (0-29.7 mM). Excitation wavelength was taken as 281 nm

705

and emitted in the range of 400-550 nm. The inset shows the variation of fluorescence

706

intensity of Ct-DNA bound to pirenzepine as a function of NaCl concentration. (b) Salt

707

dependence of pirenzepine binding constants. log Ka v/s log [Na+] plot.

708

Fig. 10.

709

Molecular modelling studies (a) Cluster histogram showing the possible conformations. (b)

710

Surface representation showing the docked structure of pirenzepine to 1BNA dodecamer

711

d(CGCGAATTCGCG)2 (c) Stereoview of the docked conformation of pirenzepine-Ct-DNA

712

complex showing the possibility of hydrogen bonds. (d) The formation of hydrogen bonds

713

between the O-atom of carboxyl group of pirenzepine and the –NH group of guanine base

714

pair (DG-10 of chain A) O---H: 2.8Å and hydrogen bond between the H atom of pirenzepine

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and O group of adenine base pairs (DA-17 of chain B) H---O: 2.6Å. The relative binding

716

energy of the complex system was found to be -7.5 kcal/mol. (e) Electrostatic interaction

717

involving the negative charge of phosphate backbone of Ct-DNA and pirenzepine. (f)

718

Competitive molecular docking of pirenzepine with Hoechst complexed with Ct-DNA.

RI PT

719 720 721

SC

722 723

M AN U

724 725 726 727

731 732 733 734 735 736 737 738 739

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730

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Tables

741

Table1. Iodide quenching parameters obtained in presence and absence of Ct-DNA. Ksv (M-1)

Pirenzepine

1.51x103±0.439

Without Ct-DNA

Ra

S.D.b

0.9991

0.29336

Relative reduction in Ksv (%)

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742

Pirenzepine-Ct-DNA complex

0.9996

SC

6.62

1.41x103±0.288

0.24917

a

R is the correlation coefficient. bS.D. is standard deviation.

744

Table 2. Ksv values obtained for the displacement of fluorescent dyes by pirenzepine

745

from Ct-DNA.

1.8 x 102

Hoechst

5.09 x 102

DAPI

5.37 x 102

S.D.b

0.9999

0.00548

0.9999

0.02847

0.9999

0.03723

EP

Ethidium bromide

Ra

TE D

Ksv (M-1)

Dye

M AN U

743

a

R is the correlation coefficient. bS.D. is the standard deviation.

747

Table 3. Isothermal titration calorimetry derived thermodynamic parameters for the

748

interaction of pirenzepine with Ct-DNA.

AC C

746

T (K)

303 749

K (M-1)

1.00x103

n 0.950

∆H(Kcal/mol) -2.54

∆S (cal/mol/K) 14.2

∆G(kcal/mol) -6.8

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