original research article doi: 10.26479/2020.0603.01 ...molecular docking of heroin...
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Original Research Article DOI: 10.26479/2020.0603.01
MOLECULAR DOCKING OF HEROIN (3,6-DIACETYL-MORPHINE)
AND BUTYRYLCHOLINESTERASE
Aditya Narayan Singh*
Indian Academy Degree College- Autonomous, Hennur Cross, Bangalore, Karnataka, India.
ABSTRACT: The non-specific serine hydrolase enzyme butyrylcholinesterase is known for
catalysis of a wide range of choline-based esters in the body. It is synthesized in the liver and is
mainly found in the blood plasma. Heroin is a narcotic opioid which is an intensively addictive
depressant. It is an analogue of morphine having two acetyl groups in its structure as compared to
morphine. Metabolism of Heroin is a two-step process where heroin is initially deacylated into 6-
monoacetyl-morphine (6-MAM) followed by another deacylation to form morphine.
Butyrylcholinesterase is an enzyme involved in the first deacylation step of heroin to 6-MAM. The
objective of this work was to visualize the binding of heroin into the active site gorge of the enzyme
and to identify the amino acids to which heroin binds in within the active site. This was carried out
using molecular docking tools Autodock vina and PyMol.
Keywords: Butyrylcholinesterase, Heroin, deacylation, active site gorge, molecular docking.
Article History: Received: March 20, 2020; Revised: April 11, 2020; Accepted: May 01, 2020.
Corresponding Author: Aditya Narayan Singh*
Indian Academy Degree College- Autonomous, Hennur Cross, Bangalore, Karnataka, India.
1. INTRODUCTION
Butyrylcholinesterase (BuChE) (EC 3.1.1.8) also referred to as pseudocholinesterase is a non-
specific serine hydrolase enzyme synthesized in the liver that is responsible for the hydrolysis of
various choline-based esters [1]. It can be found mainly in the blood plasma in human beings. The
enzyme catalyzes a broader range of substrates in contrast to another cholinergic enzyme
acetylcholinesterase (AChE). Heroin is an opioid drug which is an analogue of morphine formed by
acetylation of the two hydroxyl groups of morphine (phenolic 3-hydroxyl and alcoholic 6-hydroxyl
group) [2]. The drug is a potent depressant which is usually snorted or injected after which the users
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often report sensations of pleasure. The effect of heroin is intense in the brain cells which may stop
producing chemical signals responsible for pleasure in absence of the drug. Metabolism of heroin
have been extensively studied in animals which concluded that breakdown of heroin is a sequential
step which involves deacylation of heroin to 6-monoacetyl morphine followed by another
deacylation to yield morphine [3]. The effectivity of the metabolites of heroin is far greater than that
of heroin itself which shows a half-life of 3-5 minutes in the human blood [2]. BuChE is encoded
by BCHE gene located on chromosome 3q26 [4]. The enzymes AChE and BuChE are observed to
exhibit different tissue distributions where AChE is mainly localized in the neural synapses and
BuChE has a non-specific tissue distribution found in liver, heart, lungs and plasma. Contrary to
AChE the role of BuChE is not seen to be physiologically essential, but to be involved in breakdown
of organic compounds such as aspirin [5], cocaine [6, 7] and activation of drugs such as heroin [8].
The enzyme exhibits catalytic efficiency of 4.5 µmol/L per min [9].The active site gorge of the
enzyme consists mainly of a catalytic triad, anionic site, acyl pocket, oxyanion hole and peripheral
anionic site [10]. The amino acids which constitute the active site are [10]:
1. Catalytic triad- SER198, GLU325, HIS438
2. Anionic site- TRP82, TYR128, PHE329
3. Peripheral anionic site- ASP70, TYR332
4. Oxyanion hole- GLY116, GLY117, ALA199
5. Acyl pocket- LEU286, VAL288
Studies in recent times have observed BuChE to hydrolyze Ghrelin secreted majorly by
enteroendocrine cells to inactive desacyl ghrelin. This leads to a suppression in apetite [11]. It was
observed that inactivation of ghrelin also resulted in significant reduction of fighting tendency
among mice who had overexpression of BuChE when compared to BuChE knock-out mice [12].
BuChE is also known to play a role in Alzheimer’s Disease (AD), an irreversible neurodegenerative
disorder observed during recent researches. The enzyme is observed to stimulate the maturation of
amyloid-beta plaques which results in AD [1]. Therefore, therapy methods have been diagnosed to
inhibit the activity of BuChE which involve the use of drugs such as Donepezil, Galantamine,
Rivastigmine and Tacrine [13]. Tacrine shows liver toxicity to some extent and hence non-toxic
analogues of tacrine are used as therapeutics for AD [14]. Cymserine and its analogs N1-
norcymserine, N1-phenethylnorcymserine, N8-benzylnorcymserine, N8-norcymserin, and N1,N8-
bisnorcymserine were chemically synthesized and found to be selective inhibitors of
butyrylcholinesterase [15]. Dialkyl phenyl phosphates such as di-n-butyl phenyl phosphate, di-n-
pentyl phenyl phosphate and di-n-cyclohexyl phenyl phosphate were observed as potent inhibitors
of the butyrylcholinesterase as determined by flexible docking [16]. Molecular docking was carried
out to detect the binding of heroin within the enzyme. Molecular docking is a method in the field of
molecular modelling which allows the prediction of the orientation in which one molecule binds to
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another molecule for the formation of a stable complex [17]. The strength of association between
the two molecules is used to predict the orientation and affinity of one molecule within the other
molecule. With molecular docking the binding pattern of a drug can be used to assess the application
of that drug against a particular protein or enzyme [18] . A typical docking experiment involves
assessing the binding of a ligand to its protein based on their pairwise interaction energy [19] which
can be visualized by a simulation provided within the docking software. The most commonly used
docking software are MGL Tools, AutoDock vina (developed by The Scripps Research Institute)
[20] , Chimera and PyMol, few of which have been used to visualize the binding of heroin in
butyrylcholinesterase.
2. MATERIALS AND METHODS
1. Obtaining protein x-ray crystallography structure of butyrylcholinesterase
The x-ray crystallography structure of the enzyme butyrylcholinesterase was obtained from RCSB
Protein Data Bank using the PDB id 6esy which was isolated from the Homo sapiens [21] . The
protein was obtained as a dimer (Fig-1) bound to thioflavin-T which was selected and removed from
the structure using Autodock tools.
Fig 1: Structure of 6esy obtained by PDB
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Fig 2: X-ray crystallography structure of chain A observed in PyMol
2. Obtaining the ligand structure
The structure of the ligand used for docking – Heroin (Diacetyl-morphine) was obtained from
PubChem database using the ID 5462328. The ligand was further converted to the AutoDock
structure file (.pdbqt) using OpenBabel to be used for docking with butyrylcholinesterase.
Docking of the ligand and enzyme was carried out using AutoDock tools (version 4.2) followed by
AutoDock vina to cross verify the docking result. Prior to docking, chain B of the enzyme was
deleted and all water molecules were removed from the protein. Followed by this, polar hydrogens
were added and Gasteiger charge was computed. To minimize the docking area so as to allow
docking within the binding pocket, a 60 Å x 60 Å x 60 Å grid box was chosen as the binding site.
The grid parameters were saved as a configuration text file to be used later for Autodock vina. The
protein was set rigid and an Autogrid program was run prior to docking using default grid parameters.
The grid file was used for docking using the default parameters of Autodock tools. Post docking,
the results were analysed using the Autodock analysis tools. In case of Autodock vina, the binding
result was ranked according to its RMSD value. The result output file was analyzed using PyMol
tools and a consistent result was verified.
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3. RESULTS AND DISCUSSION
The docking result showed perfect binding of Heroin in the active site gorge of butyrylcholinesterase.
Heroin was found to establish polar contact with ASP70, TYR128 and TYR332 of lengths 3.4Å,
2.3Å and 3.3Å respectively as measured by PyMol. (Fig-3)
Fig 3: Polar bonds formed by heroin in the active site gorge
The binding pocket (surface representation) of ligand in the protein can be observed in Fig-4
Fig 4: Binding pocket of heroin in the active site gorge
The ligand was found to bind in the active site gorge of the enzyme as shown in the following
figures:
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Fig 5: Anionic site around Heroin
Fig 6: Peripheral anionic site around Heroin
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Fig 7: Oxyanion hole around Heroin
Fig 8: Catalytic triad around Heroin
With the above data obtained using molecular docking it is evident that the ligand Heroin binds
within the active site gorge of butyrylcholinesterase and therefore acts as a substrate for the enzyme.
Similar to the function of serine hydrolases, catalysis can proceed with acyl-enzyme intermediate
through the serine residue in the active site followed by hydrolysis of the intermediate to form the
free enzyme and 6-monoacetyl-morphine.
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The active site gorge of the BuChE as visualized using PyMol is given in Figure-9.
Fig 9: active site gorge of BuChE
Figure 10: Heroin in the active site gorge of BuChE
4. CONCLUSION
Butyrylcholinesterase is a non-specific serine hydrolase which is involved in the breakdown of
several choline-based esters in the body. This study was carried out with the objective of finding out
the binding of the opioid heroin in the active site of the enzyme. With the results obtained from
molecular docking it was observed that heroin was bound within the active site gorge of the enzyme.
It was bound specifically to ASP70, TYR128 and TYR332 amino acids with polar bonds. Since
heroin is able to bind within the active site gorge, it acts as substrate for the enzyme which deacylates
it to 6-monoacetyl-morphine, the latter having a more potent neural effect with respect to heroin
itself.
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ETHICS APPROVAL AND CONSENT TO PARTICIPATE
Not applicable.
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base of this research.
CONSENT FOR PUBLICATION
Not applicable.
AVAILABILITY OF DATA AND MATERIALS
The authors confirm that the data supporting the findings of this research are available within the
article.
FUNDING
None.
CONFLICT OF INTEREST
Authors have no conflict of interest.
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