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
