International Journal of Biological Macromolecules 73 (2015) 9–16 Contents lists available at ScienceDirect International Journal of Biological Macromolecules j ourna l h o mepa ge: www.elsevier.com/locate/ijbiomac Interaction of coumarin with calf thymus DNA: Deciphering the mode of binding by in vitro studies Tarique Sarwar, Sayeed Ur Rehman, Mohammed Amir Husain, Hassan Mubarak Ishqi, Mohammad Tabish ∗ Department of Biochemistry, Faculty of Life Sciences, A.M. University, Aligarh, U.P. 202002, India a r t i c l e i n f o Article history: Received 5 September 2014 Received in revised form 29 September 2014 Accepted 8 October 2014 Available online 11 November 2014 Keywords: Coumarin DNA binding Docking a b s t r a c t DNA is the major target for a wide range of therapeutic substances. Thus, there has been considerable interest in the binding studies of small molecules with DNA. Interaction between small molecules and DNA provides a structural guideline in rational drug designing and in the synthesis of new and improved drugs with enhanced selective activity and greater clinical efficacy. Plant derived polyphenolic com- pounds have a large number of biological and pharmacological properties. Coumarin is a polyphenolic compound which has been extensively studied for its diverse pharmacological properties. However, its mode of interaction with DNA has not been elucidated. In the present study, we have attempted to ascertain the mode of binding of coumarin with calf thymus DNA (Ct-DNA) through various biophysical techniques. Analysis of UV–visible absorbance spectra and fluorescence spectra indicates the formation of complex between coumarin and Ct-DNA. Several other experiments such as effect of ionic strength, iodide induced quenching, competitive binding assay with ethidium bromide, acridine orange and Hoechst 33258 reflected that coumarin possibly binds to the minor groove of the Ct-DNA. These observations were further supported by CD spectral analysis, viscosity measurements, DNA melting studies and in silico molecular docking. © 2014 Elsevier B.V. All rights reserved. 1. Introduction There has been considerable interest in the binding studies of small molecules with DNA owing to their diverse applications [1]. DNA is the pharmacological target of many drugs that are cur- rently in clinical use or are in advanced clinical trials [2,3]. DNA has been the recognition and characterization site for the interac- tion of small molecules as they yield effective information for the development of therapeutic agents for controlling gene expressions [4,5]. Studying the interaction of pharmaceutical agents with DNA is also essential for understanding their mode of action and struc- tural specificity of their binding reactions [6]. Interaction between small molecules and DNA provides a structural guideline in ratio- nal drug designing. It helps in the synthesis of new and improved drug entities with more selective activity, greater clinical efficacy and lower toxicity. Small molecules may bind to DNA double helical structures through three different modes (i) Electrostatic binding: Abbreviations: Ct-DNA, calf thymus DNA; EB, ethidium bromide; AO, acridine orange. ∗ Corresponding author. Tel.: +91 9634780818. E-mail address: tabish.bcmlab@gmail.com (M. Tabish). occurs due to interaction between negatively charged DNA phos- phate backbone and positively charged end of small molecules (ii) Intercalative binding: occurs when small molecules interca- late within stacked base pairs thereby distorting the DNA backbone conformation [7] (ii) Groove binding: occurs due to hydrogen bond- ing or van der Waals interaction with nucleic acid bases and small molecules in the deep major groove or the shallow minor groove. Groove binders cause no or little distortion of the DNA backbone [8]. However, many small molecules can directly interact with DNA, and the factors for these interactions are quite complex. Study- ing DNA as a drug target is attractive due to the availability of the genome sequence, well-studied three-dimensional DNA struc- ture and the predictability of their accessible chemical functional groups. However, the number of known DNA-based drug targets is still very limited as compared to the protein-based drug targets [9]. Coumarin (1,2-benzopyrone), the parent molecule of coumarin derivatives, is the simplest compound (Fig. 1A) of a large class of naturally occurring polyphenolic substances made of fused ben- zene and apyrone rings [10]. Coumarin is present in a wide variety of plants including cassia, lavender, yellow sweet clover, tonka beans, green tea, woodruff and in fruits such as bilberry and cloud- berry. Coumarins have recently attracted much attention because of their broad pharmacological properties. Coumarin has been http://dx.doi.org/10.1016/j.ijbiomac.2014.10.017 0141-8130/© 2014 Elsevier B.V. All rights reserved.