BINDING EFFICACY AND ELUCIDATION OF QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP OF ACETANILIDE AND ITS DERIVATIVES WITH BOVINE SERUM ALBUMIN AND THEIR INHIBITION AGAINST COX1HTML Full Text
BINDING EFFICACY AND ELUCIDATION OF QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP OF ACETANILIDE AND ITS DERIVATIVES WITH BOVINE SERUM ALBUMIN AND THEIR INHIBITION AGAINST COX1
V. Violet Dhayabaran*1, S. Aishwarya 1 and R. Renganathan 2
Department of Biotechnology and Bioinformatics, Bishop Heber College 1, Tiruchirappalli-17, Tamil Nadu, India
Department of Chemistry, Bharathidasan University 2, Tiruchirappalli-36, Tamil Nadu, India
Serum albumins are the most abundant proteins in plasma with many physiological functions. Among them, BSA has a wide range of functions involving the binding, transport and delivery of fatty acids, porphyrins, bilirubin, steroids, etc and it is home to specific binding sites for metals, pharmaceuticals and dyes. Recently, nanotechnology has become a popular term in the current science and technology. Nanotechnology has been introduced for the food and drug industry, including encapsulations and delivery systems. BSA nanoparticles were prepared and their binding efficacy with the available analgesics such as acetanilide and its derivatives were studied. The value of apparent rate constant Kapp from the interaction between acetanilide and BSA by UV visible spectroscopic and fluorescence technique was found to be 2.294X106. The quenching rate constant of BSA-Acetanilide was found to be 1.0345X1015M-1 S-1. There are two binding sites in BSA for acetanilide. A QSAR study was performed for the different analgesics. Inhibition of Acetanilide and its derivatives with the Cyclooxygenase (COX 1) was studied using docking mechanism. The electro chemical behavior of acetanilide is studied and it is found to be reversible.
in the current science and technology. It refers to the research at the scale of 100nm or less. Nanotechnology has been introduced for the food and drug industry, including encapsulations and delivery systems. In drug carrier systems, the particles should be small enough to penetrate through the capillary bed. Bioactive ingredients such as nutrient, phytochemicals, nutraceuticals and drug particles can be incorporated into nanoparticles to maximize delivery efficiency and desirable benefits 1. Serum albumins are the most abundant proteins found in plasma and are found to have many physiological functions such as transport, delivery of fatty acids, porphyrins, bilirubin, etc and it has specific binding sites for metals, drugs and dyes 2.
The binding properties of BSA and drugs are being investigated recently and nanoparticles of BSA can be prepared to enhance the drug delivery. Drug delivery is one of the important roles of nanotechnology. For application of nanoparticles in drug industry, different nanosized carriers have been developed such as nanoparticles, surface modified nanoparticles and solid lipid nanoparticles. Nanoparticles are commonly prepared by emulsification or desolvation methods 3. The desolvation methods for the preparation on nanoparticles of size 100 nm was prepared by adjusting pH of the solutions 4.
Analgesics are the drugs which relieve pain by acting on the central nervous system and they reduce pain without loss of consciousness. Analgesics can be divided into two main groups namely: Narcotic analgesics and Non- narcotic analgesics 5. Many important properties of molecules are dependent on the microscopic properties from which the molecules are built up. The complex relationship between the molecules and their properties occur in their physicochemical and biomolecular properties as well as in material behavior, in biochemical methods such as molecular docking and analogues design 6, 7. Quantitative Structure Activity Relationship (QSAR) is a method which correlates molecular structure to its in vitro or in vivo biological properties. Computational virtual screening approaches assess the interaction of the small molecules with the macromolecular target in silico 8.
The present work is focused on studying the binding efficacy of acetanilide with BSA and to elucidate the Quantitative Structure Activity Relationship of acetanilide. The work was also aimed to investigate the inhibitory action of acetanilide against the Cyclooxygenase protein (CoX1) based on molecular docking mechanism.
MATERIALS AND METHODS:
Reagents and Chemicals: The chemicals, acetanilide and cinnamic are purchased from Aldrich of high degree of purity (98%), Bovine Serum Albumin (96%). Fatty acid free product (No. A8806, Sigma) was dissolved in double distilled water to prepare stock solution of (1X10-4) which was then stored at 0-4oC. All measurements were performed at room temperature.
Fluorescence Spectroscopy: Fluorescence Spectroscopy or fluorometry is a type of electromagnetic spectroscopy which analyses fluorescence from sample using a beam of light most probably ultra violet rays. It is the technique used for structural elucidation and compound identification. These instruments record the response of samples by three parameters namely the number of bands in the spectrum, the position of the bands and intensities of the bands at all wavelength simultaneously.
Cyclic Voltammetric Studies: Cyclic Voltammetry is used to study the electrochemical properties of an analyte in a solution. The electrode potential is ramped linearly versus time and ends when a set potential is reached. The working electrode potential ramp is inverted. The current at the working electrode is plotted versus the applied voltage to give the cyclic voltammogram trace.
UV- Visible and Fluorescence Studies: UV-VIS spectrometry is an instrumental technique that provides ideal methods for the determination of micro and semi micro quantities of analytes in a sample. The UV-VIS spectrum arises due to the transitions amongst the electronic energy levels of the absorbing species. 0.023g of BSA is dissolved in 5ml of water. The concentration is calculated by measuring the absorbance at 280nm. 3ml solution of BSA containing the concentration of 1X10-5 M was prepared from 1X10-4 M stock solution and titrated by successive additions of 30 µl stock solution of acetanilide (1X10-3 M). UV-Visible spectra of all the solutions were recorded in UV-Visible range. Fluorescence spectra were then measured at ambient temperature.
Docking Studies: Docking studies of the acetanilide derivatives with the target protein was carried out using the software Argus lab. The crystal structure of Cyclooxygenase – 1(PDBID-3N8Z) was downloaded from the Protein Data Bank (PDB). The structures of acetanilide and its derivatives were downloaded from Pubchem, a database for small organic molecules. The compounds were docked with the target at its active site. The energy values and interaction of the compounds with the target were determined and the best inhibitor compound was selected based on the energy values and interaction.
QSAR Studies: To elucidate the Quantitative Structure Activity Relationship of the acetanilide and its derivative compounds DRAGON software was used. The downloaded structures of the compounds were loaded into the software and the constitutional and molecular descriptors were calculated.
RESULTS AND DISCUSSIONS:
Interaction between acetanilide and BSA by UV Visible Spectroscopic and Fluorescence Technique: Table 1 shows the absorption spectrum of BSA in water and water containing acetanilide at different concentrations. In presence of the quencher the absorbance of BSA is increased markedly without change in the location of the peak (280nm). This inference is due to the reason that while adding the quencher solution to BSA solution, the BSA molecules gets absorbed on the surface of acetanilide and involved in the formation of ground state complex of the type BSA-Acetanilide. The newly formed complex also has the absorption at 280nm. This is the reason for increase in absorption of BSA with the addition of acetanilide. This result indicated that there is an interaction between acetanilide and BSA and it is existed through ground state complex formation. The value of apparent association constant Kapp was determined from the plot and it is 2.294 x 106.
TABLE 1: ABSORPTION OF BSA IN PRESENCE AND ABSENCE OF ACETANILIDE AT 280nm
Fluorescence quenching of BSA by Acetanilide: The effect of increasing concentration of acetanilide on the fluorescence emission spectrum of BSA was recorded (Table 2). Addition of acetanilide to the solution of BSA resulted in the quenching of its fluorescence emission. Io and I are the emission intensities of BSA and Q is the concentration of the quencher. A linear plot is obtained when Io/I is plotted against [Q]. The quenching rate constant is determined and it is found to be Kq as 1.0345 x 1015 M-1 S1. Fig. 6 shows Stern- Volmer plot of the fluorescence quenching of BSA (1x10-6M) acetanilide.
TABLE 2: EFFECT OF FLUORESCENCE QUENCHING OF BSA BY ACETANILIDE
|Acetanilide x 105||X 10-5 M λ||I||Io/I|
Binding constant and number of Binding Sites: In general, the quenching constant Kq of various kinds of quenchers to biopolymer is 2.0 x1010. But for BSA-Acetanilide system Kq was found to be a higher and the quenching rate constant on 1.0345 x 1015 M-1 S1. This shows that the quenching of BSA by acetanilide is not dynamic in nature. Therefore, it depends on the formation of complex between Acetanilide and BSA. The calculated value of binding constant is 0.25 and the number of binding sites n is 2.0. The value of n equals 2 and thus indicates the existence of two binding sites in BSA for Acetanilide.
Mechanism of interaction between Acetanilide and BSA: The fluorescence of BSA may be due to the presence of amino-acid residue such as tryptophan and phenyl alanine. Hence, spectroscopic methods are usually applied to study the conformation of serum protein in synchronous fluorescence spectroscopy. Usually the amide I peak for BSA occurs in the region of 1600-1700cm-1 and amide II band near 1548cm-1. Fig. 2, shows the FT-IR spectra of BSA a sharp peak near 1630cm-1 appears while a broad peak appears near 1685cm-1. In Fig. 3, BSA in presence of acetanilide shows the amide peak at 1718cm-1. From the shift in peak position we conform that the conformation of BSA has been affected by the addition of acetanilide as supported by similar observation made earlier.
To elucidate the Quantitative Structure Activity Relationship of acetanilide and their derivatives: The quantiitative Structure Activity Relationship of the acetanilide and its derivatives were analyzed (Table 3). All the five compounds were found to obey the Lipinski’s rule of 5. From the results of QSAR, the compound found to satisfy all the necessary parameters to act as drug.
TABLE 3: QUANTITATIVE STRUCTURE ACTIVITY RELATIONSHIP STUDIES FOR ACETANILIDE AND ITS DERIVATIVES
|H Bond donor||Molecular weight||Log P||H bond acceptor|
|Compounds||Drug like index||Depressant 80||Psychotic 80||Infective 80||Wienner index test||Binding energy Kca/mol|
|4 chloroacetyl acetanilide||1||0||0||0||117.22||-5.116|
To study the inhibition efficiency of acetanilide and their derivatives against the protein Cox 1 based on docking studies: The 5 different derivatives of acetanilide were docked against the Cox protein (Table 4). Their interaction binding energy and bond distance were analyzed. The results of docking of the acetanilide derivatives with the Cox1 protein, suggested that the compound Acetaminophen has the lowest energy. It was also found to have the same interaction as that of the ligand. So Acetaminophen was found to inhibit the protein Cox1 with greater efficiency. Fig. 7 indicates the interaction of the different compounds with COX protein.
TABLE 4: ENERGY AND H-BOND INTERACTION TESTED FOR THE COX1 INHIBITION BY THE ACETANILIDE AND ITS DERIVATIVES USING DOCKING STUDIES
|S. No||Ligand||Energy (kcal/mol)||Interaction||Bond distance|
|1||FLP||-11.4744||527 Ala (NH….O)||2.58Ao|
|2||ACETANILIDE||-8.9164||522 Met(NH….O)||2.693 Ao|
|3||ACETAMINOPHEN||-8.616||387 Trp (NH….O)||2.490 Ao|
|527 Ala(NH….O)||2.980 Ao|
|4||BROMO ACETANILIDE||-8.589||355 Ser (OH….O)||2.771 Ao|
|353 Ser (NH….O)||2.714 Ao|
|5||ORTHO CHLOROACETANILIDE||-10.669||353 Ser(OH….O)||2.592 Ao|
To investigate the electrochemical behavior of acetanilide by cyclic voltammetry studies: Fig. 4 and 5 (Cyclic Voltammogram of Acetanilide 0 to 1V) show Cyclic Voltammogram of acetanilide which clearly indicate a well defined oxidative peak a negative -0.4 V and at more the potential at +1.2 V as reported. Furthermore it does not show the reduction peak. Therefore the electrochemical process is found to be reversible.
FIG. 1: ABSORPTION SPECTRUM OF BSA (1X10-6) IN PRESENCE AND ABSENCE OF ACETANILIDE
FIG. 2: FT-IR SPECTRUM OF BSA. Legend: BSA- Blue & BSA +Acetanilide- Brown
FIG. 3: IR OF BSA IN THE PRESENCE AND ABSENCE OF ACETANILIDE. Legend: BSA- Blue & BSA +Acetanilide- Brown
FIG. 4: CYCLIC VOLTAMMOGRAM OF ACETANILIDE
FIG. 5B: CYCLIC VOLTAMMOGRAM OF ACETANILIDE 0 to 1V
FIG. 6: STERN- VOLMER PLOT OF THE FLUORESCENCE QUENCHING OF BSA (1X10-6 M) ACETANILIDE
FIG. 7: INTERACTIONS OF (A) LIGAND FLUOROIBROFEN, (B) ACETANILIDE, (C) ACETAMINOPHEN, (D) 4 CHLOROACETYL ACETANILIDE, (D) 4 BROMO ACETANILIDE AND (E) ORTHOCHLOROACETANILIDE WITH THE BINDING SITE OF COX PROTEIN
CONCLUSION: In this current study, the interaction between acetanilide and BSA was studied by UV-Visible and fluorescence spectroscopy and reveals the fact that acetanilide has a good binding capacity with BSA. The chemical association between acetanilide and BSA is found to be strong and it is exhibited by the fluorescent studies. The number of binding sites of acetanilide is found to be 2. The mechanism of interaction reveals that only the tyrosine residue is linked with the quencher acetanilide. QSAR studies reveal that the compounds obey the Lipinski’s Rule and exhibits good drug likeliness. Acetanilide is found to inhibit Cox1 efficiently with good energy values and interaction. The electrochemical behavior of acetanilide suggests it to be reversible.
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How to cite this article:
Dhayabaran VV, Aishwarya S and Renganathan R: Binding Efficacy and Elucidation of Quantitative Structure Activity Relationship of Acetanilide and its derivatives with Bovine Serum Albumin and their inhibition against Cox1.Int J Pharm Sci Res, 2012; Vol. 3(9): 3179-3185.
V. Violet Dhayabaran*, S. Aishwarya and R. Renganathan
Coordinator, Department of Biotechnology and Bioinformatics, Bishop Heber College, Tiruchirappalli-17, Tamil Nadu, India
08 May, 2012
11 June, 2012
29 August, 2012
01 September, 2012