CYP450 Flashcards
Where are the CYP enzymes located?
- Membrane bound in the endoplasmic reticulum
How many different CYPs are there and how are they classified?
> 50 different CYPs isolated and sequenced in human genome
Classified based on amino acid sequence (primary structure)
How many CYP families are there?
12, with >40% homology. Families 1, 2 and 3 are important for drug metabolism
What are the sub-families of CYPs?
> 60% homology
e.g., Family 2 has 6 subfamilies - 2A, 2B, 2C, 2D, 2E, 2F
Each has 1- 15 members (different gene products)
Each CYP enzyme has a different molecular weight
Describe the CYP nomenclature, e.g. CYP2D6
The first Arabic number represents the family (2)
The capital letter designates the subfamily (D)
The second Arabic number refers to the individual gene (6)
–>
Usually different genes are for different animal species –
e.g. CYP2D6 (human), CYP2D1 (rat), CYP2D15 (dog)
Some CYP genes conserved across animal species -e.g. CYP2E1
What CYP enzymes are predominantly expressed in the liver?
CYP2D6, CYP3A5 amongst others
What CYP enzymes are predominantly expressed in the intestine?
Largely CYP3A and CYP2C9
How is the CYP system regulated?
Regulation via multiple mechansims, both genetic and environmental. e.g. PXR receptor regulates CYP enzymes - can change or activate receptors to change the activity of proteins
EXAMPLE:St johns wort effects the PXR receptor and changes CYP3A4 and equally the PXR changes will trigger changes in intestinal pgp
–> multiple pathways for elimination
What happens to levels of certain CYP enzymes in cancer and HIV?
Reported reduced activity of CYP2D6, CYP3A4 in cancer and HIV
What is the downregulation of CYPs mediated by?
Pro-inflammatory mediators affecting gene transcription (interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α)
What 2 steps are involved in CYP450 metabolic reactions?
- Activation of O2 (potentially dangerous)
- Oxidation of drug
DRUG + O2 + NADPH + H+ –> DRUG-OH + H2O + NADP+
What is NADPH?
Cofactor needed for CYP450 mediated reactions and activity of enzymes (donator of electrons)
What are Haemoproteins?
Component of CYP system:
- cytochrome P450
- cytochrome b5
What are flavoproteins ?
Components of CYP system:
- FP1 - NADPH-cytochrome P450 reductase (FAD and FMN)
- FP2 - NADH-cytochrome b5 reductase
*reductase facilitates the transfer of electrons
What is the CYP oxidation cycle?
Efficient process that protects the activated o2 species
How many binding sites on the CYP?
Two:
- Substrate (drug molecule): active site on protein, site of catalysis, release of hydroxylated molecule
- O2 (or CO): haeme ligand, CO binding has an absorption spectrum with a maximum at 450nm (hence the name CYP450)
Does CYP3A4 have multiple binding sites?
yes, multiple, shows steric activity
Describe the first step CYP cycle - activation of O2
- Drug molecule binds to CYP450 reductase, causing conformational changes of enzyme active site and goes from low to high spin (changes reduction potential of enzyme - more open to accept electrons)
What happens after the drug molecule binds to CYP450 reductase (second step)?
- Iron is now reduced, this form of an enzyme is ready to bind the oxygen, can also bind carbon monoxide. P450-reductase facilitates the donation of electrons
What is the third step in the CYP cycle?
- Oxygen binds and goes to a form that is ready to accept another electron (by NADPH)
What is the fourth step in the CYP cycle?
- NADPH provides another electron or another source (another flava protein), peroxyl state - active state so can be quickly protonated
What is the fifth step in the CYP cycle?
- Protonated causing release of water molecule leaving CYP450 compound 1
What is the sixth step in the CYP cycle?
- Release of oxidated drug molecule (R-OH) enzyme goes back to initial form
What is FP2?
Second reductase
What does FP2 use?
- NAD not NADP
- cytochrome b5 not cytochrome P450
What does cytochrome b5 use?
- FP2 not FP1
- can transfer electrons directly to cytochrome P450
*provides flexibility in electron supply and transfer
What do CYPs have that allow them bind to certain molecules for metabolism?
Sites on the active site that will bind to a particular type of molecule
What does steric hindrance do to CYP metabolism?
Degree of steric hindrance limits access of the iron-oxygen complex and therefore prevents metabolic reaction.
*o2 needs to have access to active site
What needs to be easily abstracted from the C (or N or S) atom in CYP metabolism?
Electron or hydrogen
What CYP enzymes are considered for metabolic DDI screening in drug development?
CYP1A2, -2B6, -2C8, -2C9, -2C19, - 2D6 and CYP3A
What CYP enzyme has narrow specificity?
CYP2C9
What CYP enzyme has broad and overlapping specificity?
CYP3A4
What drug substrate preference does CYP2D6 have?
Arylalkylamines (basic) with site of oxidation 5-7Å from protonated nitrogen
e.g. ecstasy and fluoxetine
What drug substrate preference does CYP2C9 have?
Neutral or acidic molecules with site of oxidation 5-8Å from H-bond donor heteroatom.
Molecules tend to be amphipathic with a region of lipophilicity at the site of hydroxylation and an area of hydrophilicity around the H-bond forming region
e.g. tolbutamide and warfarin
What drug substrate preference does CYP3A4 have?
Lipophilic and ‘bulky’, neutral or basic molecules with site of oxidation often basic nitrogen (N-dealkylation) or allylic positions
e.g. midazolam and testosterone
What are genetic polymorphisms?
existence of poor metabolisers (lack CYP2D6 gene – cannot metabolise drugs that are substrates for CYP2D6)
What does polymorphism contribute to in drug pharmacokinetcs?
Inter-individual variability (also efficacy and safety)
- Tacrolimus – CYP3A5
- Mycophenolic acid – UGT1A9
- 6-mercaptopurine - TPMT
What may 2 drugs metabolised by the same CYP do?
Compete for metabolism - drug-drug interaction potential
What is a poor metabolizer (PM)?
Lack of CYP2D6 gene
–> Higher plasma concentrations of CYP2D6 substrates compared to EMs – higher risk of side effects (e.g. fluvoxamine), may need to adjust the dose
What are the ethnic differences in frequency of allelic variants?
For example - 7% of Caucasian population are PMs
New drugs should not be exclusively metabolised by CYP2D6!
What is an extensive metabolizer ?
Normal activity
What is an ultra-rapid metabolizer? (UM)
Gene duplication:
Low plasma concentrations of CYP2D6 substrates compared to EMs – likely risk of therapeutic failure. (e.g. paroxetine, SSRIs) may need to increase doses in these individuals
