Original Research Article DOI: 10.26479/2020.0603.01 ...MOLECULAR DOCKING OF HEROIN...

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2020 May – June RJLBPCS 6(3) Page No.1

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






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






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.






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:






Fig 5: Anionic site around Heroin

Fig 6: Peripheral anionic site around Heroin






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.






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.






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.

REFERENCES

1. Ha ZY, Mathew S, Yeong KY. Butyrylcholinesterase: A Multifaceted Pharmacological Target

and Tool. Current Protein and Peptide Science. 2020;21(1):99-109.

2. Kamendulis LM, Brzezinski MR, Pindel EV, Bosron WF, Dean RA. Metabolism of cocaine and

heroin is catalyzed by the same human liver carboxylesterases. The Joournal of Pharmacology

and Experimental Therapeutics. 1996;279(2):713-7.

3. Elliott H, Parke K, Wright J, Nomof N. Actions and metabolism of heroin administered by

continuous intravenous infusion to man. Clinical Pharmacology and Therapeutics.

1971;12(5):806-14.

4. Dalmizrak O, Teralı K, Yetkin O, Ogus IH, Ozer N. Computational and experimental studies on

the interaction between butyrylcholinesterase and fluoxetine: implications in health and disease.

Xenobiotica. 2019;49(7):803-10.

5. Masson P, Froment M-T, Fortier P-L, Visicchio J-E, Bartels CF, Lockridge O.

Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-

related neutral esters. Biochemica et Biophysica Acta. 1998;1387(1-2):41-52.

6. Mattes C, Lynch T, Singh A, Bradley R, Kellaris P, Brady R, et al. Therapeutic use of

butyrylcholinesterase for cocaine intoxication. Toxicology and Applied Pharmacology.

1997;145(2):372-80.

7. Zhan C-G, Zheng F, Landry. Fundamental reaction mechanism for cocaine hydrolysis in human

butyrylcholinesterase. Journal of the American Chemical Society. 2003;125(9):2462-2474.

8. Šagud I, Maček Hrvat N, Grgičević A, Čadež T, Hodak J, Dragojević M, et al. Design, synthesis

and cholinesterase inhibitory properties of new oxazole benzylamine derivatives. Journal of

Enzyme Inhibition and Medicinal Chemistry. 2020;35(1):460-7.






9. Salmon AY, Goren Z, Avissar Y, Soreq H. Human erythrocyte but not brain acetylcholinesterase

hydrolyses heroin to morphine. Clinical and Experimental Pharmacology and Physiology.

1999;26(8):596-600.

10. Bajda M, Więckowska A, Hebda M, Guzior N, Sotriffer CA, Malawska BJ. Structure-based

search for new inhibitors of cholinesterases. International Journal of Molecular Sciences

2013;14(3):5608-32.

11. Yao J, Yuan Y, Zheng F, Zhan C-G. Unexpected Reaction Pathway for butyrylcholinesterase-

catalyzed inactivation of “hunger hormone” ghrelin. Scientific Reports. 2016;6:22322.

12. Chen VP, Gao Y, Geng L, Parks RJ, Pang Y-P, Brimijoin S. Plasma butyrylcholinesterase

regulates ghrelin to control aggression. PotNAoS. 2015;112(7):2251-6.

13. Shuo Zhoua YY, Fang Zhenga, Chang-Guo Zhana. Structure-based virtual screening leading to

discovery of highly selective butyrylcholinesterase inhibitors with solanaceous alkaloid

scaffolds. Chemico-Biological Interactions. 2019;308:372-6.

14. Przybyłowska M, Kowalski S, Dzierzbicka K, Inkielewicz-Stepniak I. Therapeutic Potential of

Multifunctional Tacrine Analogues. Current Neuropharmacology. 2019;17(5):472-90.

15. Yu Q-s, Holloway HW, Utsuki T, Brossi A, Greig NH. Synthesis of novel phenserine-based-

selective inhibitors of butyrylcholinesterase for Alzheimer's disease. Journal of Medicinal

Chemistry. 1999;42(10):1855-61.

16. Law K-S, Acey RA, Smith CR, Benton DA, Soroushian S, Eckenrod B, et al. Dialkyl phenyl

phosphates as novel selective inhibitors of butyrylcholinesterase. Biochemical and Biophysical

Research Communications. 2007;355(2):371-8.

17. Lengauer T, Rarey M. Computational methods for biomolecular docking. Current Opinion in

Structural Biology. 1996;6(3):402-6.

18. Kitchen DB, Decornez H, Furr JR, Bajorath J. Docking and scoring in virtual screening for drug

discovery: methods and applications. Nature Reviews Drug Discovery. 2004;3(11):935-49.

19. Feig M, Onufriev, Alexey, Lee, Michael S, Im, Wonpil, Case, David A, Brooks III, Charles L.

Performance comparison of generalized born and Poisson methods in the calculation of

electrostatic solvation energies for protein structures. Journal of Computational Chemistry.

2004;25(2):265-84.

20. O. Trott AJO. Autodock Vina: Imrproving the speed and accuracy of docking with a new scoring

function, effecient optimization and multithreading. Journal of Computational Chemistry.

2010;31:455-61.

21. Rosenberry TL, Brazzolotto X, Macdonald IR, Wandhammer M, Trovaslet-Leroy M, Darvesh

S, et al. Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase

and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study. Molecules.

2017;22(12).


Original Research Article DOI: 10.26479/2020.0603.01 ...MOLECULAR DOCKING OF HEROIN...

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