Part:BBa_K1064004

cyp450 3A4

Introduction

CYP3A4 gene is a coding protein CYP that has a function to metabolize drugs, toxin, etc in the cell naturally [18].

(Team USP-Brazil's Contribution)

It is part of the Cytochromes P450 superfamily, found in a great diversity of organisms [9] and located on the chromosome 7 in humans [15].

This enzyme is the most abundant xenobiotic-metabolizing cytochrome P450 isoform [1] being predominantly produced in the liver (95%) and small intestine [5][15] and responsible for around 30% to 50% of the metabolism of pharmaceuticals commonly used [6][15]. One of its importance relies especially in the capacity to metabolize a wide array of toxic substances, like insecticides (Azinphos-methyl; carbaryl; chlorpyrifos; diazinon; imidacloprid; parathion; phorate), insect repellents (DEET) and herbicides (Acetachlor; alachlor; ametryne; atrazine; butachlor; terbuthylazine; terbutryne) [7]. The specificity of the CYP3A4 molecule for substrates is low, and two or more ligand molecules can bind to the enzyme’s active site (heme pocket) simultaneously [1]. A number of experiments reported that CYP3A4 interacts with various ligands with non-Michaelis-Menten type kinetics [1].

Interactions and metabolization

Figure 1 : Factors that influence CYP3A4 activity. Adapted from [15]. Access on october 9th, 2021.

The CYP3A4 enzyme can have as substrate numerous drugs such as Erythromycin, alprazolam, felodipine, simvastatin, testosterone, testosterone, verapamil, vincristine among many others [11,12]. The hydroxylation of an sp³ C-H bond is one of the ways in which CYP3A4 affects its ligand [13].

A great example of interaction is with Imidacloprid (IMI). It is used as an insecticide for the protection of various types of crops and is one of the recurrent substrates of CYP3A4 [8]. As soon as the enzyme interacts with IMI, a reaction starts in which a hydroxylation of imidacloprid occurs and this can be transformed into trans-5'-hydroxyl-IMI or cis-5'-hydroxyl-IMI. These reaction products sometimes turn out to be less toxic than the initial substrate [8].

Induction of CYP3A4 can be done through a variety of ligands. These ligands bind to a specific receptor called pregnane X (PXR). The activated complex forms a heterodimer that binds to a regulatory region of the CYP3A4 gene and this interaction can result in increased transcription of the enzyme due to the cooperative interaction with the proximal promoter regions of the gene [14].

[8] shows that the amino acid residues Arg192, Phe195, Ile349, Ala285, Phe284 and Phe88 are an important part of the binding of imidacloprid to the CYP3A4 molecule. [7] also shows that most of the metabolism of this neonicotinoid insecticide happens in microsomes in the liver.

Figure 2: Simulation (molecular dynamics) of binding with CYP3A4 and imidacloprid adapted from [8]. Access on october 9th, 2021.

CYP3A4 can also bind to various inhibitors, such as ketoconazole, cimetidine and ritonavir-like inhibitors [17]. Below is an image that illustrates this process.

Figure 3: Distinct molecules linked to CYP3A4 and seen from the front. Adapted from [10]. Access on october 4th, 2021.

Structure

For teams aiming to use this molecule, its ligand-free crystal structure has been reported [4] and is available to download in .pdb format at this RCSB PDB database page, with a resolution of 2.05 Å. For a CYP3A4 bound to metyrapone and progesterone, [3] has produced crystal structures that can be found at the RCSB PDB database under the pubmed ID 15256616.

A vital part of its ability to metabolize toxic substances lies on the heme group present inside its structure, which is very flexible and is able to interact with various types of substrate [1], being capable of oxidizing a wide range of structurally diverse drugs, including antineoplastic, analgesic, hormonal and immunosuppressant agents [2]. The ligand-accessible volume of CYP3A4 is estimated as 520 Å, and in the interaction between the CYP molecule and small or large substrates, the distribution pattern of hydrophobic regions, such as the Phe-cluster region, seems to be an important factor, as well as the active site volume [1].

Genetic variability

Genes that code for the CYP3A subfamily (CYP3A4, CYP3A5, CYP3A7 and CYP3A43) are located in a cluster on chromosome 7 and contain 13 exons in their structures [15,18].

It was reported that some amino acid deletions on the CYP3A4 molecule seem to be related to a drastic structural change during ligand binding [1] and rising evidence has pointed out that genetic variants in CYP3A4 greatly support interindividual variability of metabolic activity [15]. It was also identified a complete lack of CYP3A4 production in a single person due to homozygous presence of a premature stop codon in exon 9 [16].

Only a few single-nucleotide polymorphisms (SNPs) are known to influence CYP3A4 enzyme expression or function [15], and the Pharmacogene Variation Consortium (https://www.pharmvar.org/) contains 35 CYP3A4 variations described as of October 4th, 2021, available at https://www.pharmvar.org/gene/CYP3A4. The group of wild-type CYP3A4*’1 alleles (available at link above) consist of 19 subtypes, from which 18 SNPs do not affect mRNA expression or complementary DNA sequence. When analysing the variants, one intronic variant (rs35599367) has been associated with decrease in the activity of CYP3A4 and two SNPs cause loss-of-function mutations [15] while another called rs2740574 did not seem to be as influential.

Besides intronic alterations as the ones presented above, several other genetic variations were observed in the exonic region as reported in http://www.cypalleles.ki.se [15]. Indeed, most of them presented insignificant influence on CYP3A4 activity. However, others such as rs55785340 diminished theclearance of several drugs metabolized by the CYP3A4 such as midazolam, testosterone and nifedipine in in vitro systems[15].

To summarize, several variations in CYP3A4 gene have been reported which either cause a decrease or did not affect activity of CYP3A4 at all [15,18]. However, missense mutations resulting in null alleles are very rare, which points out to the importance of this enzyme for the functioning of the organisms as a whole [18].

References

1. Ohkura K, Kawaguchi Y, Watanabe Y, Masubuchi Y, Shinohara Y, Hori H. Flexible structure of cytochrome P450: promiscuity of ligand binding in the CYP3A4 heme pocket. Anticancer Res. 2009 Mar;29(3):935-42. PMID: 19414330.
2. Guengerich FP: In: Cytochrome P450: Structure, Mechanism and Biochemistry. Ortiz de Montellano PR (ed.), New York, Plenum, 1995.
3. Williams PA, Cosme J, Vinkovic DM, Ward A, Angove HC, Day PJ, Vonrhein C, Tickle IJ and Jhoti H: Crystal structures of human cytochrome P450 3A4 bound to metyrapone and progesterone. Science 305: 683-686, 2004.
4. Yano JK, Wester MR, Schoch GA, Griffin KJ, Stout CD and Johnson EF: The structure of human microsomal cytochrome P450 3A4 determined by X-ray crystallography to 2.05-Å resolution. J Biol Chem 279: 38091-38094, 2004.
5. Ding X, Kaminsky LS. Human extrahepatic cytochromes P450: function in xenobiotic metabolism and tissue-selective chemical toxicity in the respiratory and gastrointestinal tracts. Annu Rev Pharmacol Toxicol 2003;43:149–73.
6. Hellum, Bent H., and Odd Georg Nilsen. "In vitro inhibition of CYP3A4 metabolism and P‐glycoprotein‐mediated transport by trade herbal products." Basic & clinical pharmacology & toxicology 102.5 (2008): 466-475.
7. Honda, Hideo, Motohiro Tomizawa, and John E. Casida. "Neonicotinoid metabolic activation and inactivation established with coupled nicotinic receptor-CYP3A4 and-aldehyde oxidase systems." Toxicology letters 161.2 (2006): 108-114.
8. Zheng, Mei Lin, et al. "Theoretical insights into imidazolidine oxidation of imidacloprid by cytochrome P450 3A4." Journal of Molecular Graphics and Modelling 80 (2018): 173-181.
9. Lamb DC, Lei L, Warrilow AG, et al. The first virally encoded cytochrome p450. J Virol. 2009;83(16):8266-8269. doi:10.1128/JVI.00289-09
10. Samuels, Eric R., and Irina F. Sevrioukova. "An increase in side-group hydrophobicity largely improves the potency of ritonavir-like inhibitors of CYP3A4." Bioorganic & medicinal chemistry 28, no. 6 (2020): 115349.
11. Tornio, Aleksi, and Janne T. Backman. "Cytochrome P450 in pharmacogenetics: an update." Advances in Pharmacology 83 (2018): 3-32.
12. Kenworthy, K. E., J. C. Bloomer, S. E. Clarke, and J. B. Houston. "CYP3A4 drug interactions: correlation of 10 in vitro probe substrates." British journal of clinical pharmacology 48, no. 5 (1999): 716.
13. Meunier, Bernard, Samuel P. De Visser, and Sason Shaik. "Mechanism of oxidation reactions catalyzed by cytochrome P450 enzymes." Chemical reviews 104, no. 9 (2004): 3947-3980.
14. Ratajewski, Marcin, Aurelia Walczak-Drzewiecka, Anna Sałkowska, and Jarosław Dastych. "Aflatoxins upregulate CYP3A4 mRNA expression in a process that involves the PXR transcription factor." Toxicology letters 205, no. 2 (2011): 146-153.
15. Werk, Anneke Nina, and Ingolf Cascorbi. "Functional gene variants of CYP3A4." Clinical Pharmacology & Therapeutics 96.3 (2014): 340-348.
16. Werk, A. N., et al. "Identification and characterization of a defective CYP3A4 genotype in a kidney transplant patient with severely diminished tacrolimus clearance." Clinical Pharmacology & Therapeutics 95.4 (2014): 416-422.
17. Sy, Sherwin K., et al. "Modeling of human hepatic CYP3A4 enzyme kinetics, protein, and mRNA indicates deviation from log-normal distribution in CYP3A4 gene expression." European journal of clinical pharmacology 58.5 (2002): 357-365.
18. Sata, Fumihiro, et al. "CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12: evidence for an allelic variant with altered catalytic activity." Clinical Pharmacology & Therapeutics 67.1 (2000): 48-56.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 555
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1105