L17-Methods used in drug metabolism studies Flashcards
(43 cards)
what are the main cells of liver responsible for metabolism
parenchymal cells
what are the gold standard for liver studies and why?
primary hepatocytes
retain in vivo-like functions
limitations of using hepatocytes
- limited liver cell sources availible
- cryopreservation can cause loss of enzyme function
culturing:
Long-term culture leads to dedifferentiation, losing functionality.
what helps maintain function with hepatocytes ?
co-culture with non-parenchymal cells
- 3D culture with ECM sandwich mimics in vivo conditions
what are microsomes?
fragments of cell membranes containing enzymes like CYPs and UGTs essential for drug metabolism
+ve of microsomes
-cyropreservation - cna be frozen without loosing enzyme function - long term storage
pooled (multiple doners) vs induvidual (single doner)
- microsomes can easiliy be derived from any organ
why might activity levels vary in bank of human liver microsomes?
- genetic variation (SNP)
- lifetime exposure to inducers
- lifestyle differences
what does the liver S9 fraction contain ?
- cytosolic and microsomal fractions
(similar phase 2 and 2 enzymes as hepatocytes)
benefit of S9 fractions
more representivive compared with microsomes and cytosolic fractions
what is useful for investigating the contributions of individual rCYPs to metabolism ?
heterologous recominant systems
what is required for P450 expression?
- full length cDNA for the isoform of interest availible
- NADPH-P450 reductase - cytochrome B5 must be resent for activity
transient expression explain
- disapears over time
- cDNA in nucleus but doesnt achive genomic integration
- gene not reproduced
- short period of expression
SHORT TERM EXPERIMENTS
describe stable expression
- expression maintained throughout cell lineage
- genomic integration of cDNA
- gene inherited through mitosis
- daughter cells express product
GOOD FOR LONG TERM EXPERIMENTS
whats required for optimising P450 expression in e coli?
- modify cDNA N-terminal sequence
- change the amino acid after the start (Met) to Ala
- increase AT content at the 5’ end
- add histidine (his) tag at the 3’ end for easy purification
what are the outcomes of E. coli optimisation ?
- active enzyme levels are very variable
- removing part of n-terminus can make P450 enzyme soluble
- helps improve expression and funciton in e. coli
benefits of yeast as a P450 expression sytem 1
- natural (endogenous) P450 oxidoreductase
- some stains can express human oxidoreducatase
- can perform posttranslational modifications
benefits of yeast as a P450 expression sytem 2
- contains membrane organelles (human cells - good for enzyme folding)
- can isolate P450-containing microsomes for further use
- cheap and scaleable
issues with P450 expression in E.coli
- no internal membrane system
- p450s have nothing to anchor into
issues with P450 expression in yeast
- yeast already express own P450s
- difficult to isolate / investigate
benefits of insect cells compared to e coli or yeast
- carry out more complex post - translational modifications than bacteria or yeast
- cotrasnfect P450 and oxidoreductase
- HIGH LEVEL , transient expression
- allows microsome isolation
- Supersomes = ready-to-use, commercial microsomes from these cells
issues with inset cells
- baculoviral vestors are technically demanding to work with
- higher cost longer duration
What non-P450 can readily be expressed in E.coli ?
sulfotransferases, GST
what non-p450 metabolic enzyme more difficult to be expressed ?
UGT and FMO but like P450s now expressed in
insect cells using baculovirus
usefulness and benefits of p450 expression in mammalian cells
- effects of xenobiotics on cell survival
- assesment of mutagenicity
- more human relevant
- ensures proper protien folding , postranslational modification and localisation
- similar control pathways e.g. DNA repair cell cycle control gene reg
