DEFINITION
Cytochrome P450 2J2 is a protein that in humans is encoded by the CYP2J2 gene.
This gene encodes a member of the cytochrome P450 superfamily of enzymes. The cytochrome P450 proteins are monooxygenases which catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids. This protein localizes to the endoplasmic reticulum and is thought to be the predominant enzyme responsible for epoxidation of endogenous arachidonic acid in cardiac tissue.
P450 2J2 is one of the 57 P450 isoenzymes found in human and is the predominant human P450 arachidonic acid epoxygenase. Human CYP2J2 is mainly expressed in intestine and is the only CYP enzyme expressed in the cardiovascular system, including endothelium and myocardiocytes with, low expression level in the liver. This CYP enzyme is able to catalyze many reactions involved in drug metabolism and synthesis of cholesterol, steroids, and other lipids (Structural Basis for the Mutation-Induced Dysfunction of Human CYP2J2: A Computational Study. 2013).
Of most importance, CYP2J2 can adopt arachidonic acid as a major substrate in cardiac tissues and produces eicosatrienoic acids (EETs). EETs are important intracellular messengers in vascular tissues, as they play important roles in the regulation of vascular tone, haveanti-inflammatory and anti-fibrinolytic properties and protect endothelial cells from ischemic or hypoxic injuries.
fig.1 Cytochrome P450 pathways of arachidonic acid metabolism
In recent years, CYP2J2 and its EET metabolites have also been implicated in the pathologic development of human cancers for both solid tumors and hematologic malignancies. Very recent data suggest that CYP2J2 promotes the neoplastic phenotype of carcinoma cells and may represent a novel biomarker and potential target for therapy of human cancers.
THE GENE
CYP2J2 gene is localized on the 1 chromosome (location Chromosome 1 :59,893,308-59,926,790).
Official Symbol: CYP2J2.
CHEMICAL STRUCTURE AND IMAGES
A detailed understanding of the active site topology and substrate specificity requires an accurate threedimensional structure of the protein, however the 3D structure of P450 2J2 has not been determined yet. Fortunately, crystal structures of several mammalian P450s like 2R1 have been resolved and could be used as templates for homology modeling. These known P450 structures have common secondary structural elements and similarity at a gross level, although they come from diverse origins and exhibit low sequence identities between different families (Structural Basis for the Mutation-Induced Dysfunction of Human CYP2J2: A Computational Study. 2013).
In addition, their structural folds share a similar series of helices and sheets. The available crystal structure of P450 2R1 in complex with vitamin D2 (PDB entry code 3CZH) provides a good template to construct 3D models of P450 2J2 using homology modelling methods.
Primary structure
The primary sequence of human P450 2J2 (accession number P51589) has the sequence identity greater than 40% with P450 2R1 (44%). P450 2R1 has a specific vitamin D 25-hydroxylase activity since it converts vitamin D into 25-hydroxyvitamin D (calcidiol), which is the major circulatory form of the vitamin. It’s a dimeric protein consisting of Chain A and Chain B and shows two molecules of 2-hydroxypropyl-β-cyclodextrin bound at the interface between the chains to stabilize the dimer. Only P450 2R1 Chain A was used as template for homology modelling. Human P450 2R1 (chain A) crystal structure (2,3 Å resolution) shows a Query coverage of 87%, a low Expectation value (3 e -127) and has 44% sequence identity compared to the target P450 2J2.
The sequence alignment of CYP2J2 target sequence with CYP2R1 template sequence was generated using Bioinfo Genotoul.
fig.2 Sequence alignment of P450 2J2 with P450 2R1. Red colour indicates high consensus level, Blue colour indicates low consensus level.
Secondary structure
Phyre2 service was applied to predict the secondary structure of P450 2J2 using 2R1 as template. The predicted secondary structure has a good alignment with the template secondary structure. Moreover six catalytic residues are conserved from CYP2J2 to CYP2R1: E314, T315, T216, F441, C448, E457. Among these residues Cys448 represents the fifth ligand of the iron ion of the heme, that is the P450 cofactor. As expected, the secondary structural model of CYP2J2 exhibits a typical CYP folding structure with a sequence of 12 alphahelices (A, B/B’, C, D, E, F/F’, G, H, I, J/J’, K, L) and betasheets (β1, β2, β3).
fig.3 Prediction of P450 2J2 secondary structure using Phyre2.
Tertiary structure
fig.4 Structure of CYP2J2
The three-dimensional structural model of the CYP2J2 exhibits a typical CYP folding structure: 12 alpha-helices (designated from A to L) and 3 beta-sheets (designated from β1 to β3) on one side of the structural model with the heme group buried deeply inside the core of the structural model. According to the sequence analysis of human CYP enzymes, the CYP2J2 exhibits comparatively high sequence similarities with CYP2R1 (42.7%), CYP2A6 (38%), and CYP2E1 (37%).
p<>. Among the helices, C, E, F, I, and L helices constitute the core region of the protein. These helices together with B′, F′, and K helices form the active site pocket for the unsaturated fatty acid substrates, which are quite hydrophobic and much smaller than the other mammalian CYP enzymes. The volume of the active site pocket for the CYP2J2 is quite similar to those of CYP2R1 and CYP2E1, which is also reported to be involved in the fatty acid metabolism.
Six catalytic residues are conserved from CYP2J2 to CYP2R1: E314, T315, T216, F441, C448, E457. Among these residues Cys448 represents the fifth ligand of the iron ion of the heme, that is the P450 cofactor (Structural Basis for the Mutation-Induced Dysfunction of Human CYP2J2: A Computational Study. 2013).
fig.5 Overview of catalitic site of CYP2J2
Protein Aminoacids Percentage
CYP 2J2
CYP 2R1
fig.6 Protein Aminoacids Percentage of CYP2J2 and CYP2R1
SYNTHESIS AND TURNOVER
mRNA synthesis
The CYP2J2 gene has five transcripts (splice variants) :
The coding mRNA has no post-translational modification.
CELLULAR FUNCTIONS
Cellular Localization
fig.7 Subcellular location of CYP2J2
Subcellular locations of CYP2J2 are mainly the Endoplasmic reticulum and Golgi apparatus.
Biological Function
This enzyme metabolizes arachidonic acid predominantly via a NADPH-dependent olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids. One of the predominant enzymes responsible for the epoxidation of endogenous cardiac arachidonic acid pools (Structures of cytochromes P450 enzymes, in Cytochrome P450: structure, mechanism, and biochemistry. 2004).
This protein acts as an enzyme. It is known to catalyze the following reaction:
RH + reduced flavoprotein + O 2 ⇄ ROH + oxidized flavoprotein + H 2 O
It requires heme group as cofactor.
The principal molecular functions are:
- Arachidonic acid 11,12-epoxygenase activity
- Arachidonic acid 14,15-epoxygenase activity
- Arachidonic acid epoxygenase activity
- Aromatase activity
- Heme binding
- Iron ion binding
- Linoleic acid epoxygenase activity
This enzyme is involved in different biological processes:
- Epoxygenase P450 pathway
- Icosanoid metabolic process
- Linoleic acid metabolic process
- Regulation of heart contraction
- Xenobiotic metabolic process
DRUG-DRUG INTERACTION
The role that CYP2J2 plays in drug metabolism is not yet fully understood. Previous studies have identified a number of drugs from different disease areas that can be metabolized by CYP2J2,
including antihistamine drugs like astemizole, ebastine, terfenadine, and vorapaxar (Discovery and Characterization of Novel, Potent, and Selective Cytochrome P450 2J2 Inhibitors. 2013). In many publications, a number of structurally diverse substrates of CYP2J2 were identified, and with its rather broad substrate spectrum and unique tissue distribution pattern, it is possible that CYP2J2 can influence drug metabolism in the extrahepatic tissues, particularly the intestine, which may therefore dominate first pass metabolism for certain drugs such as ebastine and cause drug-drug interaction (DDI) in the gastrointestinal tract. Indeed, the latest guidance for industry on drug interaction studies from the US Food and Drug Administration (FDA) suggests that CYP2J2 should be considered if a new drug candidate is found to be not metabolized by the major CYPs, indicating the increasingly more recognized role of CYP2J2 in drug metabolism.
MOLECULAR DOCKING
The interest of docking method which follows the substrate from its entrance into the substrate access channel to its final positioning close to the heme, is to take into account
possible conformational changes of the protein and of substrates that may occur after the entrance of the substrate into the access channel.
CYP2J2 has been identified as the major contributor to the first pass metabolism of astemizole to a pharmacologically active metabolite O-desmethylastemizole. Astemizole is a longacting, non-sedating second generation antihistaminic drug used in the treatment of allergy symptoms, particularly rhinitis and conjunctivitis. It competes with histamine for binding at H1-receptor sites in the GI tract, uterus, large blood vessels, and bronchial muscle acting as a receptor antagonist. This reversible binding to H1-receptors suppresses the formation of edema, flare, and pruritus resulting from histaminic activity. This compound is also recognized as substrate by other multiple isoforms of P450s like CYP3A5, 3A7, 2D6 and acts both as substrate and inhibitor of CYP3A4 that is involved in the oxidation of approximately half of all the CYPs drugs (Identification of Novel Substrates for Human Cytochrome P450 2J2. 2010).
fig.8 Metabolism of astemizole by Cytochromes P450
Astemizole is localized in the active site with the catalytic Oxygen atom that faces the iron ion of the heme. Key amminoacidic residues responsible for the substrate binding were identified and reported in the following list:
Arg111, Arg117, Ile120, Phe121, Lys123, Asn124, Met128, Ser129, Trp134, Leu215, Val218, Thr219, Glu222, Trp251, Leu306, Asp307, Leu308, Phe310, Ala311, Glu314, Thr315, Ile376, Asn379, Val380, Arg446, Ile487, Thr488.
Thr315 is a highly conserved residue among all P450s located on helix I in close proximity to the site of oxygen binding and is thought to contribute to the protonation of the reduced oxygen intermediates through a proton relay from the exterior surface of the protein Thr315, is conserved at the same location in helix I of CYP2J2.
Several hydrophobic interactions were formed between astemizole and side chains of non polar residues constituting the binding pocket. (Identification of Novel Substrates for Human Cytochrome P450 2J2. 2010) revealed that VdW energy has a larger contribution to ligand binding than the electrostatic energy. This is in line with the fact that the binding pocket of P450 2J2 is mainly composed of hydrophobic residues: Ile, Trp, Phe, Val, Leu.
fig.9 Interaction of astemizole in the active site of CYP 2J2
Many of the drugs metabolized by both CYP2J2 and CYP3A4 like astemizole, when CYP3A4 is implicated in both intestinal and hepatic metabolism, the contribution of CYP2J2 in the intestine may have been overshadowed by that of CYP3A4, suggesting a minor role in overall intestine and liver metabolism. CYP3A4 commonly metabolized compounds at multiple sites, whereas CYP2J2 metabolism was more restrictive and limited, in general, to a single site for large compounds. Although the CYP2J2 active site can accommodate large substrates, it may be more narrow than CYP3A4, limiting metabolism to moieties that can extend closer toward the active heme iron.
fig.10 Flunarizine
Flunarizine is a selective calcium entry blocker with calmodulin binding properties and histamine H1 blocking activity ( Ki =0.13μM). It is effective in the prophylaxis of migraine, occlusive peripheral vascular disease, vertigo of central and peripheral origin, and as an
adjuvant in the therapy of epilepsy. It is a substrate of cytochrome P450 2B6, 2D6, 2C9, 1A1, 1A2 and 2A6. Flunarizine has CYP2J2 IC50 values 10-fold more selective against all the major metabolizing CYP’s inhibitor ( IC50 =0.94 mM); moreover is a not substrates of CYP2J2 and shows no time-dependent inhibition toward CYP2J2. Flunarizine is a competitive inhibitor and binds directly within the active site of CYP2J2 with the F atom right on top of the heme Fe ion, presumably blocking substrate binding. The binding pocket is also formed primarily by hydrophobic residues, largely from N-terminal loop and helix A, sheet β4 and associated loop, K/β1-4 segment, B/C segment, helix F, and helix I and the heme porphyrin ring; flunarizine is in close contact with both heme and helix I (Discovery and Characterization of Novel, Potent, and Selective Cytochrome P450 2J2 Inhibitors. 2013).
Key amminoacidic residues responsible for the inhibitor binding were identified and reported in the following list:
Arg111, Arg117, Ile120, Phe121, Leu215, Val218, Thr219, Glu222, Trp251, Leu254, Leu306, Asp307, Phe310, Ala 311, Thr315, Ile376, Val380, Pro381, Arg446, Ile487, Thr 488
fig.11 Interaction of flunarizine in the active site of CYP2J2
The binding energy between flunarizine and CYP2J2 protein is 8.900 Kcal/mol; moreover, flunarizine makes one hydrogen bond to the protein with Ile487 backbone. In the structural models, flunarizine occupies the same catalytic binding site of CYP2J2 as the substrate +_astemizole_, where it makes interactions with both the heme moiety and residues on the long helix I that are close to the catalytic center. Furthermore, the F atom on flunarizine is very close to the heme catalytic Fe atom and in the same location as the astemizole methoxy group, which is known to undergo demethylation metabolism catalyzed by CYP2J2. It is plausible that flunarizine competes the substrate not only at the binding site with astemizole but also at the catalytic center for reaction.
Increase in interest of structure and role of CYP2J2 might be very helpful to find more selective and high-affinity substrates and inhibitors, to interpret or to predict the metabolism of xenobiotics (including drugs), by CYP2J2, and to discover new possible endogenous substrates of this enzyme. The knowledge on potent inhibitors of specific CYP isoforms, is critical for the clinical use of those medicines due to the DDI. Although no DDIs involving CYP2J2 have been reported in the clinic thus far, it is possible that CYP2J2 could be an important CYP isoform for DDI in the future, especially at the gastrointestinal level, because of its predominant expression in the small intestine and its rather broad and increasing substrate spectrum.
CYP2J2 and Hypertension
Hypertension is a multifactorial and polygenic disorder where several susceptible genes interact with the environmental factors. CYP2J2 plays an important role in metabolizing arachidonic acid (AA) to biologically active cisepoxyeicosatrienoic acids (EETs), which have vasodilatory, anti-infiammatory and cardioprotective effects. In animal model studies increased CYP2J2 expression was found in the spontaneous hypertension rats while additional evidence suggests that EETs plays a role in regulating blood pressure and contributing to the etiology of essential hypertension and pregnancy induced hypertension. Recent studies have focused on the contribution of functional CYP2J2 genetic variants to hypertension using population-based association studies (Evidence for Association of Polymorphisms in CYP2J2 and Susceptibility to Essential Hypertension. 2007). The CYP2J2∗7 polymorphism has been extensively studied, as well as genetic variants in other CYP2 genes. In particular, CYP2J2∗7 (G-50T), located in the proximal promoter, has been shown to have a functional role by interrupting a critical Sp1 binding site, resulting in reduced transcription factor binding, decreased CYP2J2 promoter activity in vitro, and reduced circulating levels of CYP2J2 epoxygenase metabolites in vivo. It was found the CYP2J2∗7 variant allele was protective against hypertension in Caucasians, although no association has been detected in African- Americans. Promisingly, CYP2J2∗7 might also contribute to susceptibility to coronary artery disease. The CYP2J2∗2 variant leads to reduced AA and linoleic acid (LA) metabolism compared with the wild-type. A recent study identified a novel polymorphism (G312R/CYP2J2∗8/G18892A) was identified, which results in a functional loss of enzyme catalytic activity. In other studies, it was found a new polymorphism (rs1155002), which is the first identified intronic SNP within the CYP2J2 gene that has an effect on susceptibility to essential hypertension.
Another important study has investigated the frequency of the endothelial nitric oxide synthase (eNOS, E298D) and cytochrome P450 2J2 (CYP 2J2 50 G/T) gene polymorphisms among Egyptian hypertensive subjects (Association of eNOS (E298D) and CYP2J2 (50 G/T) gene polymorphisms with hypertension among Egyptian cases. 2012). Endothelial NOS, one of the three isoforms of nitric oxide synthase, is an enzyme that in humans is encoded by the NOS3 gene. Endothelial nitric oxide is considered an important atheroprotective mediator, as defects in the generation of nitric oxide are contributed with increase in cardiovascular risk factors. Clinical studies have shown that the substitution of aspartate instead of glutamate at codon 298 of eNOS gene is implicated as an impact factor for both coronary disease and hypetension. However, there was no significant association between eNOS and CYP2J2 and hypertensionin Egyptian subjects even if it is necessary to perform a large-scale study and prospective cohort study with increased number of subjects to more accurately judge association of the polymorphism with hypertension.
