Cytochrome p450 Flashcards
How many CYP genes?
> 200 CYP genes characterised, 12 mammalian families
Evolutionary changes in location:
- Prokaryotic- cytoplasmic
- Eukaryotic- membrane bound
CYP membrane:
Endoplasmic* reticulum (microsomes):
- Exogenous* (drugs, pollutants)
- Endogenous (fatty acids, prostaglandins)
Mitochondria:
- Steroidogenesis
How many CYPs isolated and sequenced in human genome?
> 50 different CYPs
- Classified based on amino acid sequence (primary structure
How many families of CYPs?
12
> 40% homology
- Families 1, 2 and 3 important for drug metabolism
How many CYP subfamilies?
- > 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
CYP nomenclature?
e.g. CYP2D6
- 2 represents the family
- D designates the subfamily
- 6 = individual gene
Usually different genes are for different animal species –
e.g. CYP2D6 (human), CYP2D1 (rat), CYP2D15 (dog)
What does the CYP system do?
- Provides metabolic ‘safety
net’ - Acts on a wide variety of chemical structures
- Common goal to introduce/expose functional groups
- Regulation via multiple mechanisms, both genetic and environmental (prior drug and chemical exposure)
- Large inter-individual variability
CYP expression and activity in disease:
- Reported reduced activity of CYP2D6, CYP3A4 in cancer and HIV1
Downregulation of CYPs mediated by pro-inflammatory mediators affecting gene transcription interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α)
Inconsistent and limited data on the effect of obesity on CYP expression and activity2,3
Effect of coeliac disease, CKD, liver cirrhosis on certain CYPs4
Multicomponent system – operates in two steps.
1) Activation of O2 (potentially dangerous)
2) Oxidation of drug
Components of CYP system:
1) Haemoproteins
- cytochrome P450
- cytochrome b5
2) Flavoproteins
- FP1 - NADPH-cytochrome P450 reductase (FAD and FMN)
- FP2 - NADH-cytochrome b5 reductase
3) Lipid component - phosholipid membrane (endoplasmic reticulum in the intact cell)
Central role and binding properties of CYPs:
- CYP oxidation cycle
- -> Efficient, protects activated O2 species
- Two binding sites
- -> Substrate - active site on protein, site of catalysis
- O2 (or CO) - haeme ligand
- -> CO binding has an absorption spectrum with a maximum at 450nm
- Enzyme kinetics often described by Michaelis-Menten principles
- -> Assumes a single substrate binding site
- -> Rapid equilibrium and formation of Enzyme-Substrate complex
CYP cycle – activation of O2 :
1) Start with drug molecule (RH) binded to CYP450
2) Binding of molecule = conformational change from low to high spin status
3) Changed reduction potential of enzyme – more likely to accept electrons
4) The donor of electron = NADPH
5) TO FACILIATE ELECTRON TRANDFER YOU NEED FP1
6) Iron now reduced – enzyme is ready to bind to oxygen
7) The oxygen then binds
8) Ready to accept another electron (two electron transfers) – source = NADPH again
9) Peroxy state = active – will very quickly be protonated which will trigger release of water molecule
10) Cytrop450??
11) Release of oxidated drug molecule (R-OH)
12) Enzyme now goes back to initial form where the water molecule is bound
Function of other microsomal components:
- FP2 (second reductase) uses:
- -> NAD not NADP
- -> cytochrome b5 not cytochrome P450
- Cytochrome b5 uses:
–> FP2 not FP1
–> can transfer electrons directly to cytochrome P450
Provides flexibility in electron supply and transfer
Determinants of CYP metabolism:
- Topography of the active site – specificity
- Degree of steric hindrance restricting access of the iron-oxygen complex to the possible site of metabolism
- Ease of electron or hydrogen abstraction from the C (or N or S) atom
Mechanism the same for all CYPs
Specificity of CYPs:
- CYP enzymes considered for metabolic DDI screening in drug development:
- -> CYP1A2, -2B6, -2C8, -2C9, -2C19, - 2D6 and CYP3A
- -> Focus will be on three major CYPs:
- CYP2D6, CYP2C9 and CYP3A4
- Each CYP enzyme has its own degree of specificity
- > Some CYPs have narrow specificity (CYP2C9)
- > Some have broad and overlapping (CYP3A4)
- Chemical structure and phys-chem properties of drug affect the preference for a particular CYP
Guidelines for drug substrate preference:
- CYP2D6: Arylalkylamines (basic) with site of oxidation 5-7Å from protonated nitrogen
- CYP2C9: 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
- CYP3A4: Lipophilic and ‘bulky’, neutral or basic molecules with site of oxidation often basic nitrogen (N-dealkylation) or allylic positions
What is CYP2D6:
Arylalkylamines (basic) with site of oxidation 5-7 Å from protonated nitrogen
Ecstasy
Fluoxetine
What is CYP2C9?
Neutral or acidic molecules with site of oxidation 5-8 Å from H-bond donor heteroatom.
What is CYP3A4?
Lipophilic and ‘bulky’, neutral or basic molecules with site of
oxidation often basic nitrogen (N-dealkylation) or allylic positions
(a-carbons)
Importance of knowing which CYPs metabolise particular drug:
- Genetic polymorphisms – existence of poor metabolisers (CYP2D6)
- Polymorphism contributes to inter-individual variability in drug pharmacokinetics (also efficacy and safety)
- –> Tacrolimus – CYP3A5
- -> Mycophenolic acid – UGT1A9
- -> 6-mercaptopurine - TPMT
- Drug-drug interaction potential
- -> 2 or more drugs metabolised by the same CYP may compete for metabolism
Multimodal distribution of CYP2D6 in population:
- Poor Metaboliser (PM) – lack CYP2D6 gene
- Extensive Metabolizer (EM)
- Ultra-rapid metabolizer (UM)
Does ethnicity affect metabolism:
- For example - 7% of Caucasian population are PMs
- New drugs should not be exclusively metabolised by CYP2D6!
Therapeutic consequences of CYP2D6 polymorphisms:
PM
- Higher plasma concentrations of CYP2D6 substrates compared to EMs – higher risk of side effects (e.g. fluvoxamine)
Therapeutic consequences of CYP2D6 polymorphisms: Extensive Metabolizer (EM)
normal activity
Therapeutic consequences of CYP2D6 polymorphisms:
Ultra-rapid Metabolizer (UM) – gene duplication
- Low plasma concentrations of CYP2D6 substrates compared to EMs – likely risk of therapeutic failure
Predicting and avoiding drug-drug interactions:
- Important to know the contribution of CYPs to metabolism of a new drug
- What is the major CYP contributing? Multiple enzymes involved?
- Is the drug an inhibitor of CYPs/transporters?
(Routine screening - FDA requirement) - Co-administration of the enzyme inhibitor leads to increased plasma concentrations of the ‘victim’ drug
- Findings have labelling implications!
