Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs

Accepted Manuscript Title: Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs Author: Mahmoud Kandeel Abdullah Altaher Yukio Kitade Magdi Abdelaziz Mohamed Alnazawi Kamal Elshazli PII: DOI: Reference:

S1476-9271(16)30223-7 http://dx.doi.org/doi:10.1016/j.compbiolchem.2016.07.009 CBAC 6569

To appear in:

Computational Biology and Chemistry

Received date: Revised date: Accepted date:

9-5-2016 22-6-2016 25-7-2016

Please cite this article as: Kandeel, Mahmoud, Altaher, Abdullah, Kitade, Yukio, Abdelaziz, Magdi, Alnazawi, Mohamed, Elshazli, Kamal, Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs.Computational Biology and Chemistry http://dx.doi.org/10.1016/j.compbiolchem.2016.07.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Evolution of camel CYP2E1 and its associated power of binding toxic industrial chemicals and drugs Running title Camel CYP2E1

Mahmoud Kandeel1,2, *, Abdullah Altaher1, Yukio Kitade3, Magdi Abdelaziz2, Mohamed Alnazawi1, Kamal Elshazli2 1

Department of Physiology, Biochemistry and Pharmacology, Faculty of Veterinary, King Faisal

University, Alhofuf, Alahsa, Saudi Arabia; 2Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelshikh University, Kafrelshikh 33516, Egypt, 3Department of Chemistry and

Biomolecular Science, Gifu University, Japan

*Mahmoud

Kandeel

Department of Pharmacology, Faculty of Veterinary Medicine, Kafrelshiekh University, Kafrelshiekh 33516, Egypt. [email protected]

1

Graphical abstract

2

Highlights     

CYP2E1 is a molecular sieve for detoxifying small molecules Camel CYP2E1 showed higher evolution rate compared with human and other test organisms Camel CYP2E1 has higher drug binding power Camel CYP2E1 more efficiently bound with small compounds Higher evolution rate and potent chemicals and toxins binding is a form of camel adaptation

Abstract Camels are raised in harsh desert environment for hundreds of years ago. By modernization of live and the growing industrial revolution in camels rearing areas, camels are exposed to considerable amount of chemicals, industrial waste, environmental pollutions and drugs. Furthermore, camels have unique gene evolution of some genes to withstand living in harsh environments. In this work, the camel cytochrome P450 2E1 (CYP2E1) is compromised to detect its evolution rate and its power to bind with various chemicals, protoxins, procarcinogens, industrial toxins and drugs. In comparison with human CYP2E1, camel CYP2E1 more efficiently binds to small toxins as aniline, benzene, catechol, amides, butadiene, toluene and acrylamide. Larger compounds were more preferentially bound to the human CYP2E1 in comparison with camel CYP2E1. The binding of inhalant anesthetics was almost similar in both camel and human CYP2E1 coinciding with similar anesthetic effect as well as 3

toxicity profiles. Furthermore, evolutionary analysis indicated the high evolution rate of camel CYP2E1 in comparison with human, farm and companion animals. The evolution rate of camel CYP2E1 was among the highest evolution rate in a subset of 57 different organisms. These results indicate rapid evolution and potent toxin binding power of camel CYP2E1. Keywords Cytochrome P450; docking; evolution rate; binding; molecular modeling

Introduction Cytochrome P450 superfamily are mixed function oxidase catalyzes the metabolic conversion of various endogenous and exogenous substances increasing its solubility and become easily excreted [1]. Therefore, these enzymes comprise the first defense line for protection against drugs, xenobiotics, protoxicants, procarcinogens and chemicals toxicity [2]. In the human genome there is about 18 CYP families including about 57 CYP genes [3]. Within these large set of members, CYP 2E1 is the most abundant isoform of all CYPs as it comprises about 56% of the normally expressed CYPs. Furthermore, it was the most common CYP in the tested camel liver [4]. CYP2E has special importance due its power in metabolizing of xenobiotics, high production chemicals, industrial waste as ethanol, solvents, carbon tetrachloride, benzene, thiazole

4

and tetrazole, [5]. CYP2E1 can also metabolize some drugs as acetaminophen, chlorzoxazone, theophyline and volatile anesthetics as halothane, enflurane, sevoflurane, isolflurane and methoxyflurane [6]. Reaction of CYP2E1 with ethanol yields reactive oxygen species that lead to damage of liver [5]. In addition to its drug metabolic role, CYP2E1 is involved in maintaining subcellular reactive oxygen species pool and metabolism and its level is modulated in liver damage and oxidative stress conditions. Interestingly, both of the enzyme activity and expression levels of CYP2E1 in liver and kidney microsomes and mitochondria in camels were higher than other animals as goats, mice and rats indicating the high ability of camel liver enzymes in metabolizing wide variety of chemicals [7]. Camels are reared in arid environment. In order to adapt such habitat, camels evoluted unique pathways comprising rapid evolution of genes, new genotypes and phenotypes. For instance, camels can live in hot dry weather without shelter and without water for several weeks. Therefore, camels genetics include interesting evolution of genes controlling body thermoregulation and water metabolism [8-10]. Very little is known about drugs and toxicological agents metabolism in farm animals especially camels. By the increase in the industrial and petroleum works in the gulf region, camels became more exposed to industrial chemicals than before. Such changes

5

in camels living ecology could be faced by corresponding adaptation with camel encoded chemicals metabolizing enzymes. In the context, CYP2E1 is highly involved due to its unique wide spectrum binding with chemicals. Molecular modeling techniques are the gold standard in investigations of enzyme activity, drug discovery and molecular analysis of a protein target [11-17]. In this study, molecular modeling, phylogenetic, evolutionary analysis and docking studies are adopted to clarify the evolution, adaptation and toxic chemicals and drug binding properties of camel CYP2E1 and comparing it with human and animals CYP.

Methods 1- Annotation of camel genome Genomic

data

are

obtained

(http://www.ncbi.nlm.nih.gov)

from

and

protein

the

and

Arabian

genome camel

databases

genome

at

project

(http://www.camel.kacst.edu.sa). Handling of ESTs was by CLC genomics workbench version 7.5.1. About 17155 contigs were retrieved and stored as BLAST database. Further retrieving of protein sequence was from the protein bank.

2- Searching and retrieval of camel CYP2E1 CYP2E1 from human and Camel sequences were retrieved from NCBI and used to BLAST the camel ESTs database. The obtained contig with the highest similarity was retrieved and subjected for further molecular modeling and phylogenetic studies. The

6

retrieved contig was used to BLAST NCBI for getting the maximum number of similar sequences.

3- BLAST search and sequence alignment The retrieved camel sequences were used to BLAST non-redundant nucleotides databases at NCBI. All organisms are searched with expect value of 10. The top 100 hits with the lowest E value were downloaded and kept in one batch file for sequence alignment by using CLC main workbench. Sequence alignment was set to very accurate. The retrieved sequences were checked for repeats annotated to avoid multiple proteins for every host.

4- Phylogenetic and evolutionary analysis The evolution rate of camel CYP2E1 was estimated by Bayesian evolutionary analysis under relaxed phylogenetic models by using BEAST software package version 2.1.3 (Bayesian Evolutionary Analysis Sampling Tree [18, 19]). The nucleotide sequences from different hits were exported in NEXUS format by using geneious 7.1.7 software package. The BEAUti software was used to convert NEXUS file to xml format for Bayesian evolutionary analysis. Substitution rate was set for 1, gamma category count was set to 4 and the JC69 was used as a substitution model. Timing data were not provided and the branch lengths represented the substitutions per year with an assumed average of 1. Relaxed clock log normal was set for clock model at clock rate of 1 and uniform birth rate. Marcov Chain Monte Carlo (MCMC) mathematical model is used by BEAST, the length of chain was set to 10000000 and the log file is saved for tree

7

annotation by TreeAnnotator software. The produced tree is visualized and annotated by FigTree software. Examination of MCMC results were viewed by Tracer program.

5- Molecular modeling studies Requests for building molecular models for the camel CYP were submitted to SWISS Model server. The human CYP was used as a template for building the camel CYP in automated mode with templates in SWISS Model input. The obtained structure models were checked, minimized and prepared for docking studies.

6- Molecular docking studies Preparation of compounds: About 40 different compounds, proved to be binding with CYP2E1, were collected from the compounds bound to CYP2E1 crystal structures available at protein data bank, BRENDA enzymes information website and from literature (Fig. 1). The compounds were searched at PubChem database and downloaded in SDF format. The energy was minimized and optimized by Chem3D Pro, Cambridge soft and saved as MDL MOL files. Preparation of CYP2E1: A model of camel CYP2E1 was built by the automated mode of SWISS model server by using the human PDB ID 3T3Z structure as a template. The retrieved model was checked and energy minimized. The docking run was performed in Molegro Virtual Docker in slow or accurate mode. The following parameters were used during template docking: Grid resolution was 0.3 A, the binding site radium 15 A, number of runs were 10 per compound, maximal iterations were 1500 and energy threshold was set to 100. At first, the forces or pilocarpine recognition to the active site of CYP2E1 were concluded for template 8

docking. Docking run was followed by the default parameters of the software. The results were viewed and analyzed in the data modeler module of the software.

7- Statistical analysis Correlation matrix and correlation statistics were calculated by Molegro Data Modeler.

Results Sequence alignment The sequence alignment of camel, wild camel, human and some livestock CYP2E1 sequences is shown in Fig. 2. The selected sequences showed high similarity, ranging from 75.7-99.4%. Camel CYP2E1 contains the common cysteine residue for binding with heme (C451). Furthermore, alignment of sequences revealed that the camel CYP2E1 shares common ubiquitination site with that of other animals and human (residues 330-340). This indicates potential mode of camel enzyme inactivation by ubiquitination either naturally or inducible by drugs as carbon tetrachloride and ethanol, which decrease the action of CYP2E1 by induction of its ubiquitination. All major differences e.g. the range of amino acids from 209-300 are outside the drug binding site of CYP2E1. The most noticeable difference between human and camel CYP2E1 is the replacement of F205 in human active site with Y207 in the camel enzyme. This 9

replacement gives extra hydrogen bonding that might contribute to stronger binding of substrates with the camel enzyme (Fig. 3A-D). The structure of CYP2E1 with pilocarpine reveals that the major force of binding is by favourable steric interactions and electrostatic forces (Fig. 3E).

Phylogenetic analysis and gene evolution Phylogenetic analysis of camel CYP2E1 is shown in figures 4 and 5. Some selected phylogenetic parameters are represented in Table 1. Several settings were done for easier identification of the rate of evolution. These include the colours of branches, the size of branches, nodes size or the numbers wrote for each branch. Red colour indicates the high rate and the rate lowers as shown on the scale. Similarly, the thicker branch line indicates higher rate. The estimated rate of evolution was higher in camel CYP compared to human CYP (Table 1). Within a subset of 12 hits including camel human and some common livestock or wild animals (Fig. 4), camels showed the highest rate of evolution. Similarly, in a large-scale alignment and gene evolution rate assessments (Fig. 5), camel CYP2E1 showed markedly high evolution rate.

Docking results

10

The docking output is shown in Table 2. The selected parameters includes MolDock score, rerank score, ligand efficiency (L1) and ligand efficiency 3 (L3). MolDock score is the default output of docking energy. Rerank score is then used to assess the stability of binding. Following docking run, rerank of the output poses is performed. Rerank score is a modification of docking score with considerations of steric hindrance. Thus, rerank score is more computationally valuable than the docking score. L1 and L3 are modifications of scores considering the number of heavy atoms. The negative value of MolDock score indicates the binding of all compounds to both human and camel CYP2E1. Some positive value of rerank score was evident including clotrimazole with human and camel CYP2E1 and sulfaphenazole and zopiclone with camel CYP2E1 (Table 2). This indicates instable binding of these compounds with human or camel CYP2E1. Within the current subset of 40 compounds, camel CYP2E1 showed stronger binding elicited by higher docking score in 13 compounds (Fig. 6). These compounds include aniline, chlorocatechol, benzene, acrylamide, butadiene, toluene, chlorophenol, nitrocatechol, carbamates, diethylether and chloroform. In contrast, the camel CYP2E1 showed much lower score with zoplicone, fluoxetine, clotrimazole, eszopiclone, sulphaphenazole and isoniazid. These results indicate more powerful capability of

11

camel CYP2E1 to bind small drugs than the human CYP2E1. Additionally, the camel CYP2E1 has lower capacity to bind more bulky molecules. In this context, there is a strong negative correlation between the molecular weight of drugs and their MolDock score. In human CYP2E1, the correlation coefficient (r = -0.93, Fig. 7) is higher than that estimated for camel CYP2E1 (r = -0.62).

Discussion Camel tissues have multiple forms of CYPs [4, 20]. The excessive expression of CYP2E1 in camel liver tissues indicate that this enzyme is normally expressed and not under inducible expression. Owing to the importance of CYP2E1 in metabolism of various types of toxins and drugs, it is used in this study to compare the adaptation, evolution and its drug binding power in camels. CYP2E1 can be termed as molecular sieve due to its preferential binding with small toxins composed of few atoms. In comparison to human, the camel CYP2E1 showed stronger binding with various toxic chemicals (Fig. 6). Relative docking score indicates more potent binding of a set of small molecular weight compounds with camel CYP2E1, indicating expected higher efficiency of camel CYP2E1 in binding and metabolizing wide variety of chemicals, drugs, environmental pollutants and industrial waste. Aniline is a toxic organic

12

compound and was found to be a selective substrate for CYP2E1. Camel CYP2E1 showed stronger binding with aniline compared to human enzyme as evidenced by higher docking, rerank scores, L1 and L3 values. A similar higher docking score is calculated for aniline, benzene, nitrocatechol, amides, nitrophenol, butadiene, toluene and acrylamide. These compounds now constitute an increasing problem in the Gulf States due to environmental pollution and industrial waste. For instance, aniline is used in agricultural chemicals, rubber industry and polyurethane foam production. Acrylamide is porcarcinogen used extensively in industry. Over 5 years period, data from industrial wastewater in petrochemicals and refinery plants in Saudi Arabia showed 1377 violations from water pretreatment standards. The identified compounds included hydrocarbons and aromatic compounds as phenols, substituted phenols, benzene, toluene, alkanes and kitones [21]. Such pollution can directly or indirectly affecting the public health through consumption of plants or animal tissues [22]. The top most sources of pollution with organic toxins are petroleum, organic chemicals and synthetic industries [23]. The estimated high evolution rate of camel CYP2E1 as well as higher binding potency with many small toxins especially phenols and small hydrocarbons accounts for the unique ability of camel metabolic system in detoxifying dangerous materials in their surrounding environment. Interestingly, camel liver has the

13

lowest level of glutathione in comparison with goats, rats and mice. Furthermore, camel mitochondria and microsomes showed less reactive oxygen species (ROS) production, therefore, camel liver has has strong GSH-dependent redox defense mechanism for protection of mitochondria from ROS [7]. Taken together, the potent antioxidant activity in camel liver and potent drug-binding camel CYP2E1, which is also involved in maintaining subcellular ROS level and metabolism, implicate adaptation and evolution of camel CYP2E1 to manage various toxins. For further analysis of this finding, modeling studies showed the presence of extra-hydrogen bonding possibility in camel structure which contributes to the strength of binding. In addition, energy mapping of enzymes active sites revealed more favourable electrostatic forces in camel CYP2E1 (Fig 3 D), which contributes to forceful binding. Some compounds as zoplicone, fluoxetine, clotrimazole, eszopiclone, sulphaphenazole, coumarin, disulfiram and isoniazid showed lower binding to camel CYP2E1 compared to the human CYP. This finding does not exclude the binding and metabolic event. However, this suggests the expected slower metabolism of these substrates in camels. An interesting feature is the great difference in evolution rate of CYP2E1 between the wild camel and the domestic Arabian camel, 3.2 and 0.35, respectively. This might be due to the effect of domestication, which brought domestic camels in contact with wider

14

range of compounds and chemicals. The lower binding energy of disulfiram with camel CYP2E1 indicates its expected lower efficiency in inhibiting the enzyme and so that, lower efficiency in preventing the consequences of unwanted metabolism by CYP2E1 e.g. the hepatotoxic effect of halothane after anesthesia after metabolism with CYP2E1 [24]. Disulfiram is a potent inhibitor of human CYP2E1 [25], this is also is concluded in camels as it seems to has the highest binding energy among all the dataset of compounds tested in camel CYP2E1 (Fig. 6). The strongest binding substances with camel CYP2E1 are eszopiclone, sulfaphenazole, disulfiram, clotrimazole, diethyl carbamates and coumarin. This agrees with their potential inhibitory activity to CYP2E1. The anesthetic drugs halothane, enflurane, isoflurane, methoxyflurane and servoflurane are substrates from CYP2E1. The docking profile shows that the docking parameters are almost similar among all anesthetic between human and camel enzymes. Clinical trials of halothane usage in camels reveal the safety of halothane in camels as well as all events during anesthesia was predictable [26, 27]. Unfortunately, inorganic fluoride releases during metabolism of halothane causing serious toxicity [24]. In this context, the higher expression level of CYP2E1 in camels suggests its vulnerability to toxicity by fluoride during the use of these compounds as anesthetics in camels.

15

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Drummond AJ, Suchard MA, Xie D, Rambaut A: Bayesian phylogenetics with BEAUti and the BEAST 1.7. Molecular biology and evolution 2012, 29(8):1969-1973.

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Figure legends Fig. 1. The compounds used in this study Fig. 2. Similarity score and sequence alignment of camel, wild camel, pig, human, dog, cat, horse, sheep, mouse, cattle and buffalo CYP2E1. Fig. 3. The docked drugs in the active site of human (A) and camel (B) CYP2E1. Hydrogen bonds are in sky blue dashes. Electrostatic interactions are shown in C and D. The binding for pilocarpine with CYP2E1 (E) showing favourable steric interactions (blue) and hydrostatic interactions (green) with the active site. Fig. 4. Phylogenetic analysis and evolution rate values for camel, wild camel, pig, human, dog, cat, horse, sheep, mouse, cattle and buffalo CYP2E1. Fig. 5. Phylogenetic analysis and evolution rate values for camel in a large subset of organisms. Fig. 6. The results of docking score (A) by descending values according to the human CYP2E1. The relative score is shown in B (camel docking score/human docking score*100). Fig. 7. Plotting and correlation of human and camel CYP2E1 docking scores with mw of compounds.

19

Figure 1

20

Figure 2

21

Figure 3

22

23

Figure 4

24

Figure 5

25

Figure 6

26

Figure 7

27

Table 1. Some selected parameters of rate obtained by BEAST analysis and Tracer program for CYP2E1

Rate

Mutation

Tree

Camel

Cattle

Human

mean

rate

likelihood

rate

rate

rate

Large population set

0.95

0.46

-4460

3.2

0.7

0.36

Farm and companion animals

0.9

-

-14578

1.69

1.17

1.02

28

Table 2: The obtained MolDock score for docking of human and camel CYP2E1 with different compounds and drugs Human Name

MolDock

Rerank

Score

Score

camel LE1

LE3

MolDock

Rerank

Score

Score

LE1

LE3

Benzene

-13.1

-14.3

-2.2

-2.4

-16.9

-17.5

-2.8

-2.9

Ethanol

-13.2

-11.9

-4.4

-4.0

-10.5

-9.2

-3.5

-3.1

methoxyflurane

-37.4

-35.5

-3.4

-3.2

-29.2

-27.8

-2.7

-2.5

chlorzoxazone

-38.8

-32.3

-3.5

-2.9

-32.5

-24.3

-3.0

-2.2

Enflurane

-28.4

-31.8

-2.8

-3.2

-23.4

-25.4

-2.3

-2.5

Halothane

-26.3

-22.0

-3.8

-3.1

-25.3

-21.0

-3.6

-3.0

Isoflurane

-32.3

-28.8

-3.2

-2.9

-30.8

-28.1

-3.1

-2.8

methoxyflurane

-22.8

-25.2

-2.8

-3.2

-19.0

-21.5

-2.4

-2.7

4-chlorophenol

-23.7

-22.2

-3.0

-2.8

-26.7

-23.8

-3.3

-3.0

sevoflurane

-28.7

-38.0

-2.4

-3.2

-17.2

-26.6

-1.4

-2.2

Zopiclone

-82.5

-62.1

-3.1

-2.3

-26.3

78.2

-1.0

2.9

pilocarpine

-55.7

-48.4

-3.7

-3.2

-32.7

-16.7

-2.2

-1.1

Aniline

-18.5

-18.8

-2.6

-2.7

-30.2

-27.7

-4.3

-4.0

dimethylformamide

-20.5

-19.4

-4.1

-3.9

-18.8

-18.2

-3.8

-3.6

4-chlorocatechol

-26.9

-26.0

-3.0

-2.9

-39.0

-35.2

-4.3

-3.9

Eszopiclone

-92.6

-61.5

-3.4

-2.3

-45.0

-5.0

-1.7

-0.2

4-nitrocatechol

-35.4

-34.6

-3.2

-3.1

-39.3

-36.9

-3.6

-3.4

4-nitrophenol

-27.0

-27.2

-2.7

-2.7

-28.7

-26.5

-2.9

-2.7

Disulfiram

-77.2

-61.2

-4.8

-3.8

-44.1

-27.5

-2.8

-1.7

mephenytoin

-57.2

-51.1

-3.6

-3.2

-39.1

-28.7

-2.4

-1.8

sulfaphenazole

-83.3

-39.1

-3.8

-1.8

-43.0

26.2

-2.0

1.2

diethyldithiocarbamate

-37.2

-32.8

-4.6

-4.1

-40.5

-33.8

-5.1

-4.2

alpha-Naphthoflavone

-54.1

-40.4

-2.6

-1.9

-33.6

-22.2

-1.6

-1.1

Coumarin

-65.1

-55.7

-4.1

-3.5

-38.3

-32.5

-2.4

-2.0

Ethanol

-13.2

-11.9

-4.4

-4.0

-10.6

-9.3

-3.5

-3.1

Isoniazid

-28.9

-29.3

-2.9

-2.9

-15.4

-16.1

-1.5

-1.6

Toluene

-18.5

-18.8

-2.6

-2.7

-21.2

-20.4

-3.0

-2.9

acetaminofen

-37.9

-35.8

-3.4

-3.3

-29.4

-28.0

-2.7

-2.5

29

theophylline

-37.3

-37.8

-2.9

-2.9

-27.7

-25.9

-2.1

-2.0

Caffeine

-36.7

-23.0

-2.6

-1.6

-22.9

-20.5

-1.6

-1.5

clotrimazole

-74.9

43.9

-3.0

1.8

-35.5

39.6

-1.4

1.6

diethylether

-20.1

-17.5

-4.0

-3.5

-21.5

-17.8

-4.3

-3.6

fluoxetine

-54.6

-29.3

-2.5

-1.3

-22.8

-8.7

-1.0

-0.4

carbon tetrachloride

-25.5

-18.4

-5.1

-3.7

-24.5

-17.8

-4.9

-3.6

N-nitrosodimethylamine

-19.3

-19.1

-3.9

-3.8

-20.3

-19.1

-4.1

-3.8

chloroform

-19.4

-14.2

-4.8

-3.6

-19.4

-14.2

-4.8

-3.6

acrylamide

-21.4

-20.5

-4.3

-4.1

-26.1

-23.1

-5.2

-4.6

Butadiene

-14.6

-13.3

-3.6

-3.3

-16.9

-14.5

-4.2

-3.6

n-hexane

-24.0

-20.8

-4.0

-3.5

-22.7

-18.4

-3.8

-3.1

ethylcarbamate

-66.7

-57.5

-3.9

-3.4

-39.7

-36.3

-2.3

-2.1

30