Articles Bioinformatics
June 2010 Vol.55 No.18: 1877–1880 doi: 10.1007/s11434-009-3697-z
SPECIAL TOPICS:
Docking and molecular dynamics studies on CYP2D6 WANG JingFang1, ZHANG ChengCheng2 ,WEI DongQing2* & LI YiXue1* 1
Bioinformatics Center, Key Laboratory of Systems Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; 2 College of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China Received November 20, 2008; accepted December 19, 2008
Drug-metabolizing enzymes, also known as cytochrome P450s, are a superfamily of hemoglobin responsible for metabolizing more than 90% clinical drugs. Cytochrome P450 2D6 (CYP2D6) is a significant member of cytochrome P450s for the reason of metabolizing about 20% clinical drugs. In this paper, molecular docking and molecular dynamic simulations are used to investigate the active site of CYP2D6, roles of essential amino acids within the active site and time-dependent protein energy changes. The results suggest that amino acids Glu216, Asp301, Ser304 and Ala305 in the active site are likely to form hydrogen bonding interactions with substrates; the benzene ring of Phe120 and aromatic ring in the substrates form Π-Π interactions. In addition, molecular dynamics simulations prove that the catalytic conformation of CYP2D6 without ligands can be obtained by their own atomic fluctuations. The impact of ligands on protein system energy and large conformational shift is not very large. Cytochrome P450s is known for their genetic polymorphisms, which will result in severe adverse drug reactions. Ideally, we hope to use molecular modeling to investigate the differences between the substrates of wild-type and mutants while they are bonded with drugs, and predict the drug metabolizing ability of mutants. Reduce the possibility for people taking drugs that they can not metabolize, therefore reduce the rate of adverse drug reactions, and eventually establish a platform of personalized drugs to largely benefit human health. cytochrome P450, molecular docking, molecular dynamics simulations, drug-metabolizing mechanism Citation:
Wang J F, Zhang C C, Wei D Q, et al. Docking and molecular dynamics studies on CYP2D6. Chinese Sci Bull, 2010, 55: 1877−1880, doi: 10.1007/s11434-009-3697-z
The cytochrome P450s (CYPs) are a large superfamily of heme-containing enzymes responsible for drug metabolisms [1,2]. They are capable of detoxifying foreign compounds, and play the role of balancing the level of the endogenous substances [3]. These enzymes can be found mostly in the microsomes within the hepatocytes in nearly all kingdoms of life. The CYPs are well known for causing different rate of metabolism of drugs, mainly resulting from the single nucleotide polymorphism (SNPs) in the gene level [4]. The SNPs in genotypes divide human into three types of phenotypes, i.e. the poor metabolisers (PMs), the extensive metabolisers (EMs) and the ultra extensive metabolisers (UEMs). Under some circumstances, PMs and UEMs could be deadly. Therefore, it is crucial to design new drugs for *Corresponding authors (email:
[email protected];
[email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2010
different genotypes. To accomplish this goal, we must have better understanding of the structure of the CYPs’ binding pocket and the structure of the drugs which can be metabolized by certain enzyme [5–7]. In the current case, we focus on the CYP2D6, the most important enzyme in the P450 superfamily, in that it is involved in the metabolism of more than 20% of known drugs and constitutes only about 1%–2% of the entire CYP proteins. To date, some brilliant work has been done to identify the key residues of the CYP2D6 binding pocket. Asp301 and Glu216 act as recognition residues attracting ligands to the pocket [8]. Additionally, nearly all known CYP2D6 substrates contain at least one aromatic ring, which are readily to form Π-Π interactions with Phe120 and/or Phe483 in the CYP2D6 binding pocket [9,10]. Based on the crystal structure of CYP2D6 (PDB code 2F9Q) [11], dockcsb.scichina.com www.springerlink.com
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ing and molecular dynamics studies show the regular pattern of Asp301, Ala305, Glu216 and Phe120 when five similar drugs (Alprenolol, Bufuralol, Propafenone, Propranolol, and Metoprolol) are binding to the CYP2D6 binding pocket. We consider this could be significant in the personalized drug design.
1 Computational methods 1.1 Docking operations The molecular docking operations in our studies were performed by the AutoDock 3.0.3 package [12,13] to investigate the interactions between CYP2D6 and five aforementioned drug molecules. During the whole docking process, drug molecules were flexible, while the protein molecule kept rigid. AutoDock can generate a diversified set of conformations by making random changes of the coordinates of drug molecules. When a new conformation of drug molecules was generated, the search for the favorable bindings was conducted within a specified 3D docking box using genetic algorithm [14]. This approach can seek to optimize the purely spatial contacts as well as electrostatic interactions. In addition, this kind of approach was widely used to investigate the structure features of CYP2C19 [15,16], xylose reductases [17,18] as well as H5N1 influenza [19], timely providing useful structural information for both basic research and drug development in the relevant areas. 1.2 MD simulations The most favorable docking results for the six aforementioned drug molecules were further optimized by the molecular dynamics (MD) simulations. All the MD simulations in our study were employed by GROMACS 3.3.1 program in the periodic boundary conditions with GROMACS96 43a2 force fields [20]. The topology files, force field parameters, and the charges for drug molecules were generated by the online software PRODRG [21]. At the beginning, all the models were solvated in the explicit SPC water molecules. In order to neutralize the systems, several sodium ions were added to replace equal number of water molecules. Subsequently, all the models were subjected to a steepest descent energy minimization until a tolerance of 100 kJ/mol. Afterwards, the solvent molecules were equilibrated with fixed protein at 310 K for several picoseconds. Then, all the models were relaxed gradually and heated up to 310 K. Finally, 600 ps MD simulations were performed under normal temperature and normal pressure with coupling time constant of 0.1 ps and 1.0 ps. The isothermal compressibility was set to 4.5×10–5/bar for water simulations. All the MD simulations were performed with a time step of 2 fs, and coordinates were saved every 1 ps.
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Results and discussion
2.1 Molecular docking results The final docking results for the five drugs molecules are listed in Table 1. The six drug molecules have been well docked to the binding pocket of CYP2D6. The characteristics of the binding pocket play a central role for the lignad binding of CYP2D6. As shown in Figure 1, the lipophilic and hydrophilic surfaces of the binding pocket of CYP2D6 is illustration shown, in which the regions A, B, C, D and E reflect the nature and steric position of the key reisudes Phe120, Ala305, Asp301, as well as Glu216. This is also an evidence for its preferring aromic compounds: the aromic ring can form Π-Π interactions with Phe120 and other atoms such as nitrogen and oxygen atoms, and form one or more hydrogen bonds with Glu216, Asp301 and Ala305. Take the complex of CYP2D6 with metoprolol as an example to explain the interactions of drug molecules with CYP2D6, as shown in Figure 2. Phe120 is in charge of the substrate recognition and fixation. Shown by some experimental results, this residue also makes CYP2D6 have strong selectivity, though its function can be covered for Phe483 sometimes. The hydrogen bonding interactions between the substrates and CYP2D6 are mainly contributed by Glu216, Asp301 and Ala305. And the lengths and angles of the hydrogen bonds formed by drug molecules and Glu216, Asp301 and Ala305 are listed in Table 2. It is seen that Asp301(OD2) and Glu216(OE2) alternatively form hydrogen bonding interactions with the five drug molecules. The hydrogen bonds formed by these two residues have relatively shorter lengths and larger angles than those formed byother residues, which gives indication that these hydrogen bonds are stronger than others, and play important roles in the ligand binding. 2.2 MD simulations The dynamics features of the interactions between drugs
Figure 1 Illustration showing the lipophilic and hydrophilic surfaces of the binding pocket of CYP2D6.
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Table 1 List of interaction energies (kcal/mol) obtained by docking six drug molecules to CYP2D6, respectively Alprenolol Bufuralol Propafenone Propranolol Metoprolol
Einter –10.63 –10.57 –12.78 –10.66 –10.72
Etor +2.49 +1.56 +3.42 +1.87 +2.80
Ebinding –8.14 –9.02 –13.06 –10.76 –7.91
Figure 2 Illustration showing the favorable interaction of CYP2D6 with metoprolol molecule obtained by molecular docking operations using AutoDock.
Table 2 List of the lengths and angles of the hydrogen bonds formed by drug molecules and Glu216, Asp301, and Ala305, respectivelya) Asp301(O) Ala305(NH) Glu216(OE2) Asp301(OD2)
Distance (Å) Angle (°C) Distance (Å) Angle (°C) Distance (Å) Angle (°C) Distance (Å) Angle (°C)
1
2
3
4
5
2.53 78.6 2.10 138.4 1.78 16.28 – –
2.23 90.4 2.24 137.3 – – 1.84 122.7
2.17 113.1 2.30 120.7 1.89 135.5 – –
2.03 117.5 2.04 127.1 – – 1.79 141.6
2.15 117.7 2.10 120.7 – – 1.67 157.7
a) The Arabic numerals 1, 2, 3, 4, and 5 represent Alprenolol, Bufuralol, Propafenone, Propranolol, and Metoprolol, respectively.
molecules and CYP2D6 are characterized by 600 ps molecular dynamics simulations on the most favorable results after molecular docking operations. The distances of drug molecules with key residues in the binding pocket of CYP2D6 were calculated and shown in Figure 3, which can be regarded as an evidence for the results obtained in the docking processes as well.
3 Conclusions CYP2D6 is a significant member of cytochrome P450 superfamily, and is considered as the main enzymes with responsibility of metabolizing various important exogenous and endogenous compounds in many species of microorganisms, plants and animals. In human, it is related to the oxidative of 20% of all therapeutics in current clinical use-
Figure 3 The distances between drugs molecules and key residues in the binding pocket of CYP2D6 during our MD simulations.
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and has adverse drug effects, for example, enzyme induc tion and inhibition. In the current case, we have used docking operations and molecular dynamics simulations to make a detail examination of the interactions between CYP2D6 and drug molecules metabolized by CYP2D6 with the aim of gaining the insights for understanding its structural features and drug-metabolism mechanism. It is found that Phe120 and Phe483 in the binding pocket of CYP2D6 are capable of recognizing the substrates and fixing them in the binding pocket of CYP2D6. Additionally, the hydrogen bonding interactions between CYP2D6 and drug molecules are mainly contributed by Glu216, Asp301 as well as Ala305, especially the backbone oxygen atoms of Glu216 and Asp301. All these findings are in good agreement with previous experimental results. Most importantly, it is hoped that the improved understanding will provide helpful information for gaining the mechanism of drug metabolisms and personalization of drug treatment, stimulating novel strategies for finding desired personalized drugs.
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This work was supported by the National Natural Science Foundation of China (20773085 and 30870476) and National High-Tech Research and Development Program of China (2007AA02Z333).
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