L2- Drug Development And Cell Culturing Flashcards
What’s the typical pipeline for drug discovery and development starting from target identification
Lab experiments to identify targets/proteins potentiallt important for disease onset and progression
VALIDATION- first in vitro see if target could be important eg does it have proliferatice activity , then in vivo validation: is target causing tumour progression in Vivo?
CONVINCING MEDICINAL CHEMISTS- screen compounds which act against the target you’ve identified eg potential inhibitors
In vitro studies to see impact of drug
Can then be modified after various in vivo pharmacodynamic/kinetic interactions
= informed redesign where structurally optimised to specifically target or better target your target of interest
Years down the line after preclinical testing you have first phase 1 trial (you’ve tested in vivo model by this point)
What sort of in vivo mdoels can be used for both target validation and then later for pharmacodynamic/kinetic studies after sceeening compound
Mouse models or xenograft tissue
What is it called when a drug is optimised for example structurally to target your specific protein of interest after preclinical studies
Informed redesign
Give example of an experiment to use for target validation
Knock out experiments
What are the 3 considerations for targets before drug development
Is it aberrant in diseased patients eg expressed more?
Can it’s activity be targeted somehow ?
If you inhibit/ target it’s activity does this cause cell cytotoxicity (beneficial in terms of cancer development )
What is it called when chemists screen for compounds which have activity against target
High throughput screening
Compound O was found to target a protein T and then through structural modelling optimised into compound N. Scientists then compared them in vitro. How could you do this
Enzyme activity assay
See which compound has better inhibitory activity
They found compound N to be much more potent (work at better efficacy at lower concentrations). How could they test if they have cytotoxic effects
In cancer cell lines using chlonogenic cell growth assay
How are tissue cultures grown
In sterile flasks (lob sided) or incubators on growth media
what would be a appropriate cell type for this
One that is expressing protein T (cancer cell)
What is plating efficiency and how do you calculate it (%)
Number of colonies derived from started single cells (after seeding and drug treatment)
end colony number / total starting cell count
What do immortalised cells mean used for tissue culture/ chlonogenic assays often
Ones that grow / divide constantly without help to form a dense culture
Since the cells adhere to eachother and to the plastic surfaces eg flasks or plates, what needs to be done in tissue culture to separate these
Passage/split cells using trypsin to form a suspension of cells
Then counted to allow accurate replating on new growth media called seeding
What is used to count cells / ml in the suspension before chlonogenic assay and seeding of cells into new plates
Haemocytometer (has gap to put suspension in under the cover slides then put under microscope)
What does the 5x5 grid represent under microscope and what does this needed to be converted to
Cells/ 0.1microlitre Ul
Needs to be converted to /ml so you would x 10,000
What do you do with cells on the perimeter of the 5x5 grid
Count only the ones on 2/4 sides
What would you do to increase cell count accuracy
Do it three times and find mean
Why would it vary
Uneven suspension
What does 0.9% naCl mean
You have a conc of 0.9g/100ml
(W/v)
What does compound o and n need to be dissolved in first before added to chlonogenic plates now with the correct suspension diluted
Buffers
Once you gave correct buffer ingredients what do you do with it to make it liquid so you can dissolve the N and O drug in it
Dissolve in 900ml of water
Measure ph and adjust it if it’s acidic or alkali - needs to be 6.8
Make it up to final 1 litre
Would all the flasks have the same conc of drug in them
No, would have diff concentrations to determine at which one cytotoxic effects are best
What would the control in the chlonogenic assay be and why
Buffer with no drug
Make sure cytotoxic effects are because of the drug
Would you do each conc in triplicate and why would it vary
Yes
Important to know we only estimated the number of cells each plate would have , could be errors in pipetting
WhT is the colony forming efficiency for each concentration
Colonies left at the end vs cells in the beginning
Eg 216/500 cells = 43.2%
Would colony forming efficieny decrease with concentration increase
Yes
What is the IC50 and why would it be used to compare compound N and O in cytotoxicity
The conc required to kill 50% of the cells
How would you use the control to determine % of survival / colony control % by the drug
Colony number at that conc/ colony number found for control (would decrease as more colonies are dying at higher doses)
Describe the graph used to determine IC50 and why this means
Y axis would be % survival (colonies at dose/control colony number)
X axis would be different concentrations of drug
Where lines cross 50% survival is the ic50
Lower ic50 means the drug is more potent
They found using xhlonogenic assay that compound O was more cytotoxic. This contradicts the enzyme assay. What could be some explanations : explains differences between in vitro vs in vivo for drug testing
Potnetnially compound N has difficulty entering actual cells with a membrane, or could be efflux Ed out
Also potentiallt means that protein T inhibition might not be the factor causing cytotoxicity
If you have 5 playes and you want to calculate plating efficiency, whag value would you take as initial
Mean value
