In Silico Molecular Docking Studies Of Butylated Hydroxytoluene Biology Essay


In aerobic respiration, reactive oxygen species are usually formed in mammalian cells. These are molecules and ions of oxygen that have unpaired electrons making them highly reactive. ROS attack cellular structures, proteins and DNA. This damage may further lead to cancer, heart diseases and cerebrospinal diseases. Antioxidants scavenge these molecules and prevent ROS damage. In this study we aim to examine in-silico, the effect of antioxidant Butylated Hydroxytoluene (BHT), derived from marine Streptomyces on enzymes that act against ROS. The enzymes involved in ROS damage that were studied are Alpha-1-microglobulin, Superoxide dismutases, and Catalase and Thioredoxin peroxidase. The chemical structures of ligand and the macromolecule were obtained from the Protein Data Bank (PDB). The softwares AUTODOCK 4.2 and PYMOL were used to investigate the molecular interactions. Pre-designed and standardized protocol was followed and results were obtained. The specific interactions were obtained were analyzed on basis of their binding energies. The result show that Erythrocyte Catalase 1F4J showed specifically highest binding energy of -5.11kcal/mol and Cu, Zn Superoxide Dismutase 1PU0 showed lowest binding energy of -4.12kcal/mol. The docking studies show the use of BHT as a potent drug along with its antioxidant activity. The assessment studies help us to further examine the precise role of ROS damage in human health and disease, and the use of antioxidants as an alternative remedial effect.


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Reactive oxygen species and free radicals are being regularly formed in our body as by products of aerobic respiration and metabolism. In dormant state 1-2% of oxygen is ordinarily converted into superoxide contributing to the major ROS production during mitochondrial respiration [1]. Superoxide reactive molecules are know to cause mitochondrial oxidative stress and distrub normal functioning of the mitochondrion. This damage is frequently observed in many pathological conditions. ROS damage has been linked in the pathology of several human diseases, including cancer, therosclerosis, malaria, and rheumatoid arthritis and neurodegenerative diseases [2].

The presence of the triphenylmethyl radical (Ph3C·) demonstrated by Moses Gomberg in 1900, paved the way for further research of free radical and reactive molecules. A free radical is defined to be any chemical species that is capable of independentexistence and that atleast on unpaired electron in an orbital. The hydrogen atom is know to be the simplest free radical, containing one proton and one electron. The only electron being unpaired qualifies Hydrogen atom to be a free radical. (.) is usually inserted to indicate the number of unpaired electrons in a free radical. Reactive oxygen species is a combined term that largely describes O2-derived free radicals such as superoxide anion (O2•−), hydroxyl (HO•), peroxyl (RO2•), and alkoxyl (RO•) radicals, as well as O2-derived nonradical species such as hydrogen peroxide (H2O2) [3].

Reactive oxygen species (ROS) being by-products of normal metabolism are beneficial and harmful in nature. ROS species are called as redox messengers wherein they function in intracellular signaling and cellular regulation at physiologically low levels. They also are used in defense mechanisms of cells against infectious agents. Multiple systems and enzymatic reactions help in modulating ROS neutralization. Apart from these regulatory mechanisms ROS escape from the mitochondrial respiratory chain and result in oxidative stress, a process that mediate damage to cell structures, including lipids, membranes, proteins, and DNA. The excess of ROS leads to oxidative modification inhibiting protein function and induce cell apoptosis.

Cells use varied mechanisms to defend themselves and reverse the harmful effects of ROS. Antioxidants along with cellular enzymes scavenge the O2·− radicals and H2O2 molecules [2]. A molecule of hydrogen peroxide (H2O2) and oxygen (O2) are produced by two superoxide anions and the reaction is catalyzed by superoxide dismutase (SOD). In the peroxisomes of eukaryotic cells, the enzyme catalase converts H2O2 to water and oxygen, and thus completes the detoxification initiated by SOD. Glutathione peroxidase is a group of enzymes containing selenium, which also catalyze the degradation of hydrogen peroxide, as well as organic peroxides to alcohols [4]. The mitochondrion majorly contributes to intracellular production of ROS. Out of the total mitochondrial O2 consumed, 1-2% of it is redirected to the formation of ROS, mainly at the level of complex I and complex III of the respiratory chain, and this diversion is considered to be tissue and species dependent [3].

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Antioxidants are key molecules with an important role in the prevention of oxidation and cellular damage. They act by inhibiting and delaying oxidative processes. Oxidation reactions produce free radicals, which start a cascade of reactions leading to damage of cells. Antioxidants terminate the chain reactions by scavenging free radicals and intermediates, inhibiting further oxidative reactions.

In recent years research in the application of antioxidants to medical treatment is increasing. Linking of development of a human disease and oxidative stress especially in cases of cancer is the key aim of the developing research. Phenolic molecules are major class of antioxidants commonly found in plant food materials. Antioxidant that is focusing here is Butylated Hydroxytoluene (BHT). Butylated hydroxytoluene (BHT), or chemically 2,6-di- tetra-butyl-p-cresol, is a fat-soluble organic compound. It is majorly classified as an antioxidant food additive. Cosmetics, pharmaceutical drugs, jet fuels, and rubber and petroleum products also use the antioxidant activity. BHT slows down the rate of autoxidation in foods and prevents changes in the foods colour, odour and taste by reacting with free radicals. BHT being fat-soluble prevents oxidative rancidity of fats.

Figure: Structure of Butylated Hydroxytoluene (BHT)

BHT is chosen as the ligand and various antioxidant enzymes are used as macromolecules to check their interaction. The bioinformatics tool is used too study these interactions and analyze whether the antioxidant BHT has significant effect in elucidating enzymatic activity of the enzymes acting against ROS damage. Assessment and further study of oxidative damage to biomolecules by means of emerging fields like bioinformatics will help us to not only advance our understanding of the underlying mechanisms but also to facilitate the supplementation and intervention studies that are designed and conducted to test ROS damage in human health and disease.

Materials and methods:

Ligand preparation

We used the antioxidant butylatedhydroxytoluene (BHT) as the ligand for the docking studies. The molecular structure was taken from NCBI and seen in CORINA online demo software and the molecule was downloaded as a pdb file.

Target Enzymes as Macromolecules

Enzymes that act against ROS damage such as alpha-1-microglobulin, superoxide dismutases, catalases, glutathione peroxidase and peroxiredoxins were selected as target macromolecules for docking studies. From the PDB database, the molecular structures of these proteins were downloaded in pdb format. The proteins being dimers, trimers or tetramers, consist of A, B, C chains. The A chain was considered for docking process suggesting for a comparative study further. Heteroatoms, ANISOU sequences and water molecules were removed and the molecules were prepared for docking.


AUTODOCK 4.2 software was used for the docking studies. It is molecular modeling and simulation software that performs docking in 2 main programs.

AutoGrid which pre-calculates grids over the target protein

AutoDock which performs effective protein-ligand interaction

Further analysis of the Protein Ligand interactions is observed in another software called PYMOL. This Software gives us 3D view of the interactions helping us to draw productive conclusions on the Protein Ligand interactions.


AUTODOCK 4.2 suite as the molecular docking tool, operating in the LINUX operating system was used with the standard protocol for docking. This software performs docking to explore the conformational states of a flexible ligand and its interaction with the enzymes. The ligand and the macromolecules are prepared for docking analysis. The number of non-polar hydrogen and rotatable bonds were calculated for the ligand using AUTODOCK4.2 software. The ligand is geometrically placed in different 3D receptor sites till an effective interaction is made. Further a semi-empirical desolvation force feild is employed to obtain the binding free energy of the interactions. The docking helped to identify the number of hydrogen bond interactions. The binding energies were identified by a number of interactions that are defined by the user, based on the genetic algorithm used. Each docking experiment consisted of 10 docking runs and analysis of the binding was done by obtaining docked conformation with minimum binding energy.










Gln11, Gln11


Cu,Zn Superoxide Dismutase



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Lys9, Val148


Manganese superoxide




Ile137, Ile137


Erythrocyte catalase



Asn369, Pro368, Pro391


Thioredoxin Peroxidase B



Val50, Phe49


Figure1: Interaction between BHT and 1EM1.

Figure 2: Interaction between BHT and 1F4J

Figure 3: Interactions between BHT and 1PUO

Figure 4: Interactions between BHT and 1QMV

Figure 5: Interactions between BHT and 3QKG

Results and Discussion:

The 3D view obtained from the PYMOL software showed the interacting sites of the ligand and the macromolecules at specific sites. The ligand butylated hydroxyl toluene was showing interaction with macromolecule alpha1-microglobulin 3QKG at sites Gln11 and Gln11 as in seen in Figure 5. In case of Superoxide dismutases 1PU0, 1EM1 interactions were observed at Val148, Lys9, Ile137, and Ile137 as observed in the Figures1&3 respectively. With the Erythrocyte catalase 1F4J interactions were observed at three residues of the macromolecule Asn369, Pro368 and Pro391as seen in Fig 2. Thioredoxin Peroxidase B 1QMV shows interaction at residues Val50, Phe49 of the macromolecule from the Fig4. In further analysis, the ligand BHT showed hydrogen bonding at positions 8th and 12th in the oxygen moiety present in the BHT ligand chosen for the study. Cu, Zn Superoxide Dismutase 1PU0 is the only molecule that shows interactions at both these loci. This result shows that erythrocyte catalase 1F4J showed highest binding energy of -5.11 kcal/mol and Cu, Zn Superoxide Dismutase 1PU0 showed lowest binding energy of -4.12 kcal/mol. High binding energy would result in increased effect of the ligand molecule on the enzyme, while lower binding energy would indicate decreased effect of the ligand.

The antioxidant activity of Butylated Hydroxytoluene was observed only in vitro as per review of previous work [2] [10]. This study just identified the definite interaction and molecules involved in the bonding of the ligand BHT and the enzymatic proteins involved in ROS damage, using in silico modeling and simulation. The involvement of the electronegative oxygen atoms in the ligand for the hydrogen bond formation and the specificity of the interaction leading to the antioxidant activity of the ligand BHT was observed in this study.


From these results we identified that the hydrogen bond interactions showed oxygen as a conserved moiety involved in binding pattern of the amino acid residues with respect to the enzymatic proteins adopted for the insilico molecular docking approach. This suggests that the ROS damage may be controlled by the use of antioxidants in unification with enzymes used. Our results substantiate the use of insilico molecular methods for the design of novel antioxidant to prevent ROS damage in cellular metabolism that could pave way for treatment of various diseases.