Lecture 4 (Assay development) Flashcards
What criteria should an assay meet?
Relevance
Reliability
Practicality
Feasibility
Automation
Cost
What is meant by ‘practicality’ of an assay?
quality and quantity of the results shall justify the investment of time, costs, and efforts
What is meant by ‘reliability’ of an assay?
results are reproducible and statistically significant
What is meant by ‘relevance’ of an assay?
readout should be unequivocally related to the target
What is meant by ‘feasibility’ of an assay?
assay can make use of available resources
What do biochemical assays mostly used?
multicolor luminescence or fluorescence-based
reagents are often developed to mimic or label the target
What are the 2 main assay types?
In Vitro/Cell-based Assays
In Vivo/Animal Models
Give some examples of In Vitro/Cell-based Assays?
- cytotoxicity assay
- cell growth
- reporter gene assay
What kinds of changes do cell-based functional assays detect?
Changes in cell morphology, cell migration,
apoptosis
In Vitro/Cell-based assays are generally more robust and cost-effective, thus often chosen for HTS.
True or False
True
In transgenic animals, certain genes are _________, _________ or __________ .
deleted, modulated, added
Give an example of a disease for which adequate models for disease do not exists
hepatitis C
In Vitro/Cell-based assays often monitor a surrogate readout, what could the surrogate readout be?
- catalytic action of an isolated enzyme
- Binding of an antibody to a defined antigen
- Growth of an engineered cell line
What are the agents involved in In vitro/Cell0based assays?
- Recombinant reagents
- Reagents that were isolated from lysates
- Whole cell crude lysates
- Intact cells
What are the advantages of In Vitro based assays over In Vivo assays?
- Robust and cost-effective
- Fewer ethical implications than whole animal experiments
- Often chosen for high-throughput screening
What kind of animal models are used in In Vivo assays?
- transgenic animals
- surgical models
- behavioural models
With the constraints of the animal model, false negatives are a great concern where a potentially useful drug is not detected.
True or False
True
What is the man aim of an assay design?
The aim is to minimize the number of handling steps and, where possible, process samples in parallel.
What kind of assays are becoming more popular?
Homogenous non-separation assays (mix and read)
Give some examples of homogenous non-separation assays (mix and read) ?
- Scintillation proximity
- Fluorescence
- Time-resolved fluorescence
- Fluorescence polarization
Explain the principle of scintillation proximity.
- solid scintillator material coated onto microbeads or wells
- scintillant-coated beads or wells are capture radioactive samples
- emitted radiation excites the scintillator –> emission of light (scintilation)
Extracellular molecular signals are usually found at very low concentrations.
What concentration?
10^-8 M or less
What is a receptor?
A receptor is any cellular macromolecule to which a drug or hormone binds to initiate its effect
What is an acceptor?
Structures which bind a ligand without evoking a native biological response
In order to interact with the ligands in a highly specific manner, what properties must receptors have?
- sensitivity
–> able to detect drug/hormones at low conc - selectivity
–> responses are elicited by narrow range of chemical substances - specificity
–> response of cells of any given type is always same
What are some ‘conditions’ of ligand binding?
-Should be target-cell specific
-Should be saturable
-Should be specific for the ligand (stereoisomers)
-Should be of high affinity
How are ligands classified?
agonists and antagonists
What is an agonist ligand?
An agonist binds to the receptor and activates that receptor
What is an antagonist ligand?
An antagonist binds to the receptor but does not activate the receptor
What is ‘receptor that is constitutively active’?
a receptor that is active in the absence of an agonist –> a situation seen with some oncogenes
What is utilised in radioligand binding assays?
cellular membrane preparations derived from animal organs or cell cultures
What is the advantage of using cellular membrane preparations that are stored at low temperature for radioligand binding assay?
Inter-assay variation is low
–> allowing the acquisition of comparable data over an extended period of time.
What are classical receptor binding and radioimmunoassays (RIA) based on?
competition between a radiolabeled tracer and the test compound for the
binding domain of a target (e.g., receptor)
What is the principle behind using ‘competition between a radiolabeled tracer and the test compound for the
binding domain of a target’ in receptor binding and radioimmunoassays (RIA)?
An active compound reduces the amount of radioligand bound to the target, and its affinity for the site can be quantified.
List out the steps in a typical ligand binding experiment
- add radioactive ligand to membrane
- allow equilibration
- separate pellet and supernatant by rapid filtration and washing and determine radioactivity
Competitive antagonism:
In the presence of a fixed concentration of a competitive antagonist, the agonist curve shows the same _____(a)_____ as in the absence of the antagonist. However, concentrations required to produce any given level of effect are _______(b)_____.
(a) maximal effect
(b) increased
Non-competitive antagonism:
High concentrations of agonist _____(a)______ completely overcome the antagonism and maximal agonist response _____(b)______ be obtained.
Why?
(a) cannot
(b) cannot
Because in non-competitive antagonism scenario, the non-competitive antagonist (ligand) is akin to non-competitive substrate that does not bind to the active site of enzyme but on a different region of the enzyme allosterically and inhibits/disables the enzyme.
What is un-competitive antagonism?
The antagonist binds to the agonist-receptor complex only and not to the free receptor.
What is a partial agonist?
Agonists that produce a lower maximal response at full receptor occupancy than do full agonists. Affinity may be greater than that of the full agonist.
Can the affinity of a partial agonist be greater than the full agonist?
Yes
What is potency?
Range of doses over which a drug produces increasing responses.
What is potency of a drug dependent on?
1.drug-receptor interaction
2. coupling efficiency to the physiological
process
3. ability to reach the relevant receptors.
What is KD?
The dissociation constant and has units of concentration (mol/dm3)
–>Concentration of ligand which, at equilibrium, causes 50% of the receptors to be occupied.
What is EC50?
Concentration of ligand at which the biological response or effect is half maximal. The EC50 of a ligand can be as much as 100-fold smaller than the KD
Functional assays generally require the use of intact cells to monitor second
messenger production or processing.
Give examples of second messengers.
- cAMP
- Ca2+ ions
Why are cellular test more cumbersome than membrane assays?
require special treatment and preparation to generate consistent data
- temperature
- sterility
- passage number
Why would cellular assays be preferred in some cases?
Cellular assays can be utilized for HTS and they provide more information than
tests that monitor only ligand affinity.
What is the technology that uses skin of Xenopus laevis to evaluate whether a test compound acts as an agonist or antagonist on expressed GPCRs?
Melanophore Technology
(immortalized amphibian melanocytes)
What is the main purpose of Melanophore Technology?
to evaluate whether a test compound acts as an agonist or antagonist on expressed GPCRs
What is the principle behind Melanophore Technology?
- melanin-containing melanosomes aggregated within the cell
- when intracellular cAMP levels exceed a certain threshold
–>melanosomes are dispersed
How do skin cells of Xenopus laevis appear visually when utilized as Melanophore Technology?
The cells appear dark when cAMP levels are high.
The cells appear light when cAMP levels are low.
What changes in the skin cell of Xenopus laevis lead to the melanosomes being dispersed?
- intracellular cAMP levels exceed a certain threshold or
- protein kinase C is activated via inositol phosphates produced by activated
phospholipase C
What does the reporter gene consist of?
promoter gene and reporter gene
Why is it important to choose an appropriate promoter for reporter gene?
The choice of promoter will ultimately control the basal level of reporter gene activity and the degree of stimulation measured.
Give an example of an endogenous promoter
c-fos
What is the downside of using endogenous promoters?
complex range of signalling events that could activate reporter gene expression and confuse the analysis of the bioassay
What is an advantage of ‘promiscuity’ of endogenous promoters?
allowing natural products that regulate different signalling and transcription processes to be identified in the same assay format
