Molecular modeling and spectroscopic studies of semustine binding with DNA and its comparison with lomustine–DNA adduct formation

Chloroethyl nitrosoureas constitute an important family of cancer chemotherapeutic agents, used in the treatment of various types of cancer. They exert antitumor activity by inducing DNA interstrand cross-links. Semustine, a chloroethyl nitrosourea, is a 4-methyl derivative of lomustine. There exist some interesting reports dealing with DNA-binding properties of chloroethyl nitrosoureas; however, underlying mechanism of cytotoxicity caused by semustine has not been precisely and completely delineated. The present work focuses on understanding semustine–DNA interaction to comprehend its anti-proliferative action at molecular level using various spectroscopic techniques. Attenuated total reflection–Fourier transform infrared (ATR-FTIR) spectroscopy is used to determine the binding site of semustine on DNA. Conformational transition in DNA after semustine complexation is investigated using circular dichroism (CD) spectroscopy. Stability of semustine–DNA complexes is determined using absorption spectroscopy. Results of the present study demonstrate that semustine performs major-groove-directed DNA alkylation at guanine residues in an incubation-time–drug-concentration-dependent manner. CD spectral outcomes suggest partial transition of DNA from native B-conformation to C-form. Calculated binding constants (K_a) for semustine and lomustine interactions with DNA are 1.53?×?10³ M^?1 and 8.12?×?10³ M^?1, respectively. Moreover, molecular modeling simulation is performed to predict preferential binding orientation of semustine with DNA that corroborates well with spectral outcomes. Results based on comparative study of DNA-binding properties of semustine and lomustine, presented here, may establish a correlation between molecular structure and cytotoxicity of chloroethyl nitrosoureas that may be instrumental in the designing and synthesis of new nitrosourea therapeutics possessing better efficacy and fewer side effects.     

Intercalation of manganese-mefenamic acid complex into double stranded of calf thymus DNA

Abstract

The interaction of the [Mn(mef)₂(phen)H₂O] complex in which mef is mefenamic acid drug and phen is 1,10 phenanthrolin ligand with calf thymus DNA (ct-DNA) was studied by using different spectroscopic methods, molecular docking and viscometery. The competitive fluorescence and UV–Vis absorption spectroscopy indicated that the complex interacted with ctDNA via intercalating binding mode with the binding constant of 1.16?×?10⁴ Lmol^?1. The thermodynamic studies showed that the reaction between the complex and ctDNA is exothermic. Furthermore, the complex induced changes in DNA viscosity. Circular dichroism spectroscopy (CD) was employed to measure the conformational changes of ctDNA in the presence of the complex and verified intercalation binding mode. The molecular modeling results illustrated that the complex interacted via intercalation by relative binding energy of ?28.45?kJ mol^?1.     

Investigating the interaction of anticancer drug temsirolimus with human transferrin: Molecular docking and spectroscopic approach

In our present study, binding between an important anti renal cancer drug temsirolimus and human transferrin (hTF) was investigated employing spectroscopic and molecular docking approach. In the presence of temsirolimus, hyper chromaticity is observed in hTF in UV spectroscopy suggestive of complex formation between hTF and temsirolimus. Fluorescence spectroscopy revealed the occurrence of quenching in hTF in the presence of temsirolimus implying complex formation taking place between hTF and temsirolimus. Further, the mode of interaction between hTF and temsirolimus was revealed to be static by fluorescence quenching analysis at 3 different temperatures. Binding constant values obtained employing fluorescence spectroscopy depicts strong interaction between hTF and temsirolimus; temsirolimus binds to hTF at 298 K with a binding constant of .32 × 10⁴ M^?1 implying the strength of this interaction. The negative Gibbs free energy obtained through quenching experiments is evident of the fact that the binding is spontaneous. CD spectra of hTF also showed a downward shift in the presence of temsirolimus as compared with free hTF implying complex formation between hTF and temsirolimus. Molecular docking was performed with a view to find out which residues are key players in this interaction. The importance of our study stems from the fact it will provide an insight into binding pattern of commonly administered renal cancer drug with an important protein that plays a pivotal role in many physiological processes.     

In vitro study on binding interaction of quinapril with bovine serum albumin (BSA) using multi-spectroscopic and molecular docking methods

The binding interaction between quinapril (QNPL) and bovine serum albumin (BSA) in vitro has been investigated using UV absorption spectroscopy, steady-state fluorescence spectroscopic, synchronous fluorescence spectroscopy, 3D fluorescence spectroscopy, Fourier transform infrared spectroscopy, circular dichroism, and molecular docking methods for obtaining the binding information of QNPL with BSA. The experimental results confirm that the quenching mechanism of the intrinsic fluorescence of BSA induced by QNPL is static quenching based on the decrease in the quenching constants of BSA in the presence of QNPL with the increase in temperature and the quenching rates of BSA larger than 10¹⁰ L mol^?1 s^?1, indicating forming QNPL–BSA complex through the intermolecular binding interaction. The binding constant for the QNPL–BSA complex is in the order of 10⁵ M^?1, indicating there is stronger binding interaction of QNPL with BSA. The analysis of thermodynamic parameters together with molecular docking study reveal that the main binding forces in the binding process of QNPL with BSA are van der Waal’s forces and hydrogen bonding interaction. And, the binding interaction of BSA with QNPL is an enthalpy-driven process. Based on Förster resonance energy transfer, the binding distance between QNPL and BSA is calculated to be 2.76 nm. The results of the competitive binding experiments and molecular docking confirm that QNPL binds to sub-domain IIA (site I) of BSA. It is confirmed there is a slight change in the conformation of BSA after binding QNPL, but BSA still retains its secondary structure α-helicity.     

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (K_a), ΔH, ΔS and ΔG values to be 15.5×10⁴M^?1, ?116.2±18.1 Kcal mol^?1, ?366 cal mol^?1K^?1 and ?7.13 Kcal mol^?1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.     

Binding affinity of pyrano[3, 2-f]quinoline and DNA: spectroscopic and docking approach

The interaction between pyrano[3, 2-f]quinoline (PQ) and calf thymus DNA (CTDNA) using spectroscopic and molecular modeling approach has been presented here. Apparent association constant (1.05×10⁵ L/mol) calculated from UV-vis specta, indicates a moderate complex formation between CTDNA and PQ. The quenching phenomena as obtained from emission spectra of ethidium bromide (EB)–CTDNA by PQ was found to be a dynamic one and the binding constants found to be 8.64, 9.25, 11.17, 12.03 × 10⁴ L/mol at 293, 300, 308, and 315 K. Thermodynamic parameter enthalpy change (ΔH) and entropy change (ΔS), indicates weak force like van der Walls force and hydrogen bonds having the key role in this binding process. The results of circular dichroism (CD) demonstrate that PQ has not induced characteristic changed in CTDNA. Results achieved from UV absorption and fluorescence spectroscopy indicating the binding mode of PQ with DNA seems to be a nonintercalative binding. The theoretical results as originating from molecular modeling showed that PQ possibly will bind into the hydrophobic region of DNA having docking binding energy = ?10.03 kcal/mol and the obtained results are in consonance with the inferences obtained from experimental data. This result is important for the better understanding of pharmaceutical aspects of binding affinity of PQ and CTDNA.     

Interaction of tRNA with Safranal,Crocetin, and Dimethylcrocetin

Abstract

Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional therapeutic. The biological activity of saffron in modern medicine is in development. Its numerous applications as an anti-oxidant and anti-cancer agent are due to its secondary metabolites and their derivatives (safranal, crocins, crocetin, dimethylcrocetin). The aim of this study was to examine the interaction of transfer RNA with safranal, crocetin, and dimethylcrocetin in aqueous solution at physiological conditions. Constant tRNA concentration (6.25 mM) and various drug/tRNA (phosphate) molar ratios of 1/48 to 1/8 were used. FT-IR and UV-Visible difference spectroscopic methods have been applied to determine the drug binding mode, the binding constants and the effects of drug complexation on the stability and conformation of tRNA duplex. External binding mode was observed for safranal crocetin and dimethylcrocetin, with overall binding constants K_safranal = 6.8 (± 0.34) × 10³ M^?1, K_CRT = 1.4 (± 0.31) × 10⁴ M^?1, and K_DMCRT = 3.4 (± 0.30) × 10⁴ M^?1. Transfer RNA remains in the A-family structure, upon safranal, crocetin and dimethylcrocetin complexation.     

Mechanistic and conformational studies on the interaction of sulfamethazine with human immunoglobulin G by molecular modeling and multi‐spectroscopic approach in vitro

Binding interaction of sulfamethazine (SMZ) with human immunoglobulin G (HIgG) has been explored under physiological conditions. The interaction mechanism was firstly predicted through molecular modeling which showed that several hydrogen bonds participated in stabilizing the SMZ ? HIgG complex. Fluorescence spectroscopy, ultraviolet–visible (UV–vis) light absorption and circular dichroism (CD) spectroscopy were used to analyze the binding site, binding constants and effects of SMZ on HIgG stability and secondary structure. The binding parameters and thermodynamic parameters at different temperatures for the reaction have been calculated according to the Scatchard, Sips and Van 't Hoff equations, respectively. Experimental results showed that the quenching mechanism was a static quenching and there was one independent class of binding site on HIgG for SMZ during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?19.12 kJ · mol^?1 and 20.22 J · mol^?1 · K^?1, respectively, which meant that the electrostatic interaction was the predominant intermolecular force in stabilizing the SMZ ? HIgG complex. Moreover, the conformational changes of HIgG in the presence of SMZ were confirmed by three‐dimensional fluorescence spectroscopy, UV–vis absorption spectroscopy and CD spectroscopy. Copyright © 2014 John Wiley & Sons, Ltd.     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma     

Molecular modeling and multi‐spectroscopic approaches to study the interaction between antibacterial drug and human immunoglobulin G

Mechanistic and conformational studies on the interaction of sulfamethoxazole (SMX) with human immunoglobulin G (HIgG) were performed by molecular modeling and multi‐spectroscopic methods. The interaction mechanism was firstly predicted through molecular modeling that confirmed the interaction between SMX and HIgG. The binding parameters and thermodynamic parameters at different temperatures had been calculated according to the Stern?Volmer, Scatchard, Sips and Van ’t Hoff equations, respectively. Experimental results showed that the fluorescence intensity of HIgG was quenched by the gradual addition of SMX. The binding constants of SMX with HIgG decreased with the increase of temperature, which meant that the quenching mechanism was a static quenching. Meanwhile, the results also confirmed that there was one independent class of binding site on HIgG for SMX during their interaction. The thermodynamic parameters of the reaction, namely standard enthalpy ΔH⁰ and entropy ΔS⁰, had been calculated to be ?14.69 kJ·mol^?1 and 22.99 J·mol^?1·K^?1, respectively, which suggested that the electrostatic and hydrophobic interactions were the predominant intermolecular forces in stabilizing the SMX?HIgG complex. Furthermore, experimental results obtained from three‐dimensional fluorescence spectroscopy, UV?vis absorption spectroscopy and circular dichroism (CD) spectroscopy confirmed that the conformational structure of HIgG was altered in the presence of SMX. Copyright © 2015 John Wiley & Sons, Ltd.     

In-vitro assessment of the binding mechanism of oxyfluorfen (herbicide) with garlic phytocystatin: multi-spectroscopic and isothermal titration calorimetric study

Abstract

Oxyfluorfen (2-chloro-1-(3-ethoxy-4-nitrophenoxy)-4-(trifluoromethyl)benzene) is a nitrophenyl ether herbicide. Phytocystatins are crucial plant proteins which regulate various physiological processes and are also responsible for maintaining protease–antiprotease balance within plants. Thus, the present article deciphers the interaction of oxyfluorfen with garlic phytocystatin (GPC) through various spectroscopic and calorimetric techniques. The cysteine proteinase inhibitory assay was done to assess the inhibitory action of GPC in the presence of oxyfluorfen. The GPC loses its inhibitory activity in the presence of oxyfluorfen. The complex formation of GPC-oxyfluorfen was shown by UV absorption spectroscopy. The intrinsic fluorescence experiment affirmed the quenching of GPC in the presence of oxyfluorfen. The Stern–Volmer quenching constant and binding constant was obtained as 6.89?×?10³ M^?1 and 9.72?×?10³ M^?1, respectively. Synchronous fluorescence showed the alteration in the microenvironment around tyrosine residues. 3D fluorescence suggested the perturbation in the polarity around aromatic residues. The isothermal titration experiment suggests that the interaction of oxyfluorfen with GPC is a thermodynamically favorable reaction. Secondary structure alteration of GPC in the presence of oxyfluorfen was studied by circular dichroism (CD). The CD result showed a reduction in the α-helical content of GPC on interaction with oxyfluorfen. Consequently, all these outcomes affirmed the formation of GPC–oxyfluorfen complex along with the structural and conformational alteration. This study identifies and signifies that the exposure of oxyfluorfen induces stress within the plant system.

Communicated by Ramaswamy H. Sarma     

Glycosaminoglycan and DNA Binding Induced Intra‐ and Intermolecular Exciton Coupling of the bis‐4‐Aminoquinoline Surfen

Despite the diverse biological activities of the glycosaminoglycan (GAG) antagonist surfen, the molecular details of its interaction with biomacromolecules remain poorly understood. Therefore, heparin and DNA binding properties of surfen were studied by circular dichroism (CD) and UV absorption spectroscopy methods. High‐affinity (K_a ~ 10⁷ M^‐1) association of surfen to the chiral heparin chain gives rise to a characteristic biphasic CD pattern due to the conformational twist of the aminoquinoline moieties around the central urea bridge. At higher drug loading, intermolecular stacking of surfen molecules alters the induced CD profile and also provokes strong UV hypochromism. In contrast to the right‐handed heparin template, binding of surfen to the left‐helicity chondroitin sulfate chains produces inverted CD pattern. Large UV hypochromism as well as polyphasic induced ellipticity bands indicate that surfen intercalates between the base pairs of calf‐thymus DNA. Extensive CD spectroscopic changes observed at higher drug binding ratios refer to cooperative binding interactions between the intercalated drug molecules. The inherent conformational flexibility of surfen demonstrated here for the first time is important in its binding to distinct macromolecular targets and should be considered for rational drug design of novel GAG antagonists. Chirality 27:605–612, 2015. © 2015 Wiley Periodicals, Inc.     

Spectroscopic Studies on the Interaction of Fluorine Containing Triazole with Bovine Serum Albumin

The binding of one fluorine including triazole (C₁₀H₉FN₄S, FTZ) to bovine serum albumin (BSA) was studied by spectroscopic techniques including fluorescence spectroscopy, UV–Vis absorption, and circular dichroism (CD) spectroscopy under simulative physiological conditions. Fluorescence data revealed that the fluorescence quenching of BSA by FTZ was the result of forming a complex of BSA–FTZ, and the binding constants (K _a) at three different temperatures (298, 304, and 310 K) were 1.516?×?10⁴, 1.627?×?10⁴, and 1.711?×?10⁴?mol L^?1, respectively, according to the modified Stern–Volmer equation. The thermodynamic parameters ΔH and ΔS were estimated to be 7.752 kJ mol^?1 and 125.217 J?mol^?1?K^?1, respectively, indicating that hydrophobic interaction played a major role in stabilizing the BSA–FTZ complex. It was observed that site I was the main binding site for FTZ to BSA from the competitive experiments. The distance r between donor (BSA) and acceptor (FTZ) was calculated to be 7.42 nm based on the Förster theory of non-radioactive energy transfer. Furthermore, the analysis of fluorescence data and CD data revealed that the conformation of BSA changed upon the interaction with FTZ.     

Underlying molecular interaction of bovine serum albumin and linezolid: a biophysical outlook

Linezolid, one of the reserve antibiotic of oxazolidinone class has wide range of antimicrobial activity. Here we have conducted a fundamental study concerning the dynamics of its interaction with bovine serum albumin (BSA), and the post binding modification of the later by employing different spectroscopic (absorption, fluorescence and circular dichroism (CD) spectroscopy) and molecular docking tools. Gradual quenching of the tryptophan (Trp) fluorescence upon addition of linezolid to BSA confirms their interaction. Analysis of fluorescence quenching at different temperature indicates that the interaction is made by static complex formation and the BSA has one binding site for the drug. The negative Gibbs energy change (ΔG⁰), and positive values of enthalpy change (ΔH⁰) and entropy change (ΔS⁰) strongly suggest that it is an entropy driven spontaneous and endothermic reaction. The reaction involves hydrophobic pocket of the protein, which is further stabilized by hydrogen bonding and electrostatic interactions as evidenced from 8-anilino-1-napthalene sulfonic acid, sucrose and NaCl binding studies. These findings also support the molecular docking study using AutoDock 4.2. The influence of this interaction on the secondary structure of the protein is negligible as evidenced by CD spectroscopy. So, from these findings, we conclude that linezolid interacts with BSA in 1:1 ratio through hydrophobic, hydrogen bonding and ionic interactions, and this may not affect the secondary structure of the protein.     

Exploring the binding mode of donepezil with calf thymus DNA using spectroscopic and molecular docking methods

Donepezil (DNP) is one of approved drugs to treat Alzheimer's disease (AD). However, the potential effect of DNP on DNA is still unclear. Therefore, the interaction of DNP with calf thymus DNA (DNA) was studied in vitro using spectroscopic and molecular docking methods. Steady‐state and transient fluorescence experiments showed that there was a clear binding interaction between DNP and DNA, resulting from DNP fluorescence being quenched using DNA. DNP and DNA have one binding site between them, and the binding constant (K_b) was 0.78 × 10⁴ L·mol^?1 at 298 K. In this binding process, hydrophobic force was the main interaction force, because enthalpy change (ΔH) and entropy change (ΔS) of DNP–DNA were 67.92 kJ·mol^?1 and 302.96 J·mol^?1·K^?1, respectively. DNP bound to DNA in a groove‐binding mode, which was verified using a competition displacement study and other typical spectroscopic methods. Fourier transform infrared (FTIR) spectrum results showed that DNP interacted with guanine (G) and cytosine (C) bases of DNA. The molecular docking results further supported the results of spectroscopic experiments, and suggested that both Pi‐Sigma force and Pi‐Alkyl force were the major hydrophobic force functioning between DNP and DNA.