Receptors And Drugs Flashcards
What must drugs show in terms of affinity for their targets?
- drugs must show a high affinity
- if a drug has a high affinity for its target then smaller concentrations of that drug will be sufficient to occupy intended targets
What do antagonists do?
They inhibit the action of agonists
Is the formation of a complex between a drug and a receptor reversible or irreversible?
They are reversible
What does occupancy mean? In terms of drugs and targets?
The number of receptors occupied by a drug.
What does the occupancy of a drug depend on?
- The affinity of the drug to the receptor
- the concentration of the drug present. Thus a higher concentration would mean more receptors are occupied
How to work out occupancy?
Number of receptors occupied / total number of receptors
What will the occupancy number range from?
Zero - one
- zero meaning that no receptor is bound to the drug and one meaning all the receptors are occupied
What does occupancy help us to understand about drugs and receptors?
It helps us to measure membrane binding and affinity
Is the response to drugs proportional to occupany?
NO!
- note drugs given can be both agonists and antagonists!
- unless the question is specifically referring to an AGONIST. In this case occupancy is proportional to affinity
- however when referring to an antagonist being the drug you’d get no clear response which would suggest binding. Therefore relying on occupancy ISNT a reliable way to find out affinity of drugs.
What is a radioligand binding assay?
This is a technique which helps us to measure the affinity of a drug which may be a ligand. (Note drugs can be ligands)
The drug is labelled so we can track its binding and effectiveness.
What are the properties of a radioligand binding assay?
- ligands needs to be radioactively labelled
- Their target can be a receptor, ion channel, enzyme, cytokine
- The ligand used can be anything like a neurotransmitter, a hormone or a cytokine.
Factors you should consider when choosing a ligand to be a radioactive ligand?
(This is in the context of testing the ligand in the lab)
- if the ligand is responsible for Non - specific binding (if it binds to anything or a specific target)
- the tissue the ligand is to be used on
- how long it needs to be incubated to work
- in experiments you need to be able to separate bound ligands from the free floating ligands
- you also need to see how effective the radioligand would be
During experiments to test affinity, how do you add radioligands and how do you remove unbound drugs?
Add radioligands to the solution with the receptors in. Make sure this is for multiple concentrations and allow the radioligand solution to equilibrate.
- once equilibrated, remove unbound drugs by filtration.
What defines a good drug?
Very little non-specific binding
EXP.1. How to find non specific binding of drugs via experimentation?
(Note drugs may bind non specifically to other things in the test tube when testing the drugs affinity, so how do we rule this out?)
- we compare non specific binding of a drug to the actual binding to a receptor.
- you measure receptor binding in two test tubes
- In the first tube you only add a radioactive drug. Note in this test tube a certain concentration of drug is used (but it isnt in excess) this means this test tube tells you non specific binding. The radioligand may bind to the receptor but also away from it like to the plastic of the test tube.
- The second has non radioactive drug in the tube but this is in excess. This causes all receptors to be occupied. The second tube tells you specific and non specific binding so it tells you total binding. (You can measure when binding has totally occurred by measuring target response as this ligand isnt radioactive. E.g. is a receptor is the target, see when receptor function has been optimised (e.g. calcium may be absorbed from solution)
- you subtract the concentration used in solution two (total bound drug) from that in solution one (non specific binding). This tells you specific binding. You subtract out non specific binding (and you know the concentration for this)
EXP.1. What is wrong with the radioligand experiement?
- The non radioactive drug and the radioactive drug must behave in the same way in both tubes
- the experiment is very time consuming
- The radioligand needs to be stable
- The ligand must be extremely pure chemically.
How to stop degradation of ligand / radioligands in solution?
- store at low temperatures (but not freezing)
- avoid light - use dark bottles
Use antioxidants - (like ascorbic acid) - use free radical scavengers (ethanol) in drug solution.
Should we use hydrogen 3 as a radiolabel?
- the labelled product is indistinguishable from native compound
- there are high specific activities obtained (greater than 80 Ci/mmol)
- there is good stability when properly stored
There is a long half life of 12.5 year
However:
- this is very dangerous to humans
Labelling is expensive and dangerous
Should we use iodine 125 as a radiolabel?
Yes!
It is safer than hydrogen
- it is cheap
It can be incorporated very well with compounds with aromatic hydroxyl groups, can be used to show high specific activities. (Greater than 2000 Ci / mmol)
Disadvantages:
It is more easily degraded than hydrogen
Has a short half life of 67 days
The idodine is a bigger molecule.
When we use tissue to test drug affinity / efficacy, we may blend isolated membrane slices.
How do we stop protein degradation from these samples?
- so note cells contain lysosomes. These have enzymes which break down proteins
- when blending these tissues, we liberate enzymes that make down the proteins we want to measure
- so we may use protease inhibitors and keep everything cold to reduce enzyme activity. This makes the experiment more reliable.
How to separate bound drugs from unbound drugs?
- this depends on the ratio of ligands binding and unbinding to targets
- these experiemnts rely on a equilibrium to be reached between this binding and unbinding
- a high affinity means the ligands bound stay and the unbound can be lost
- this separation can be done by filtration
- dialysis, column chromatography and percipitation may be done.
Again what is the equation for specific binding?
Total drug bound - non specific binding.
What affects the separation of bound drugs from unbound drugs?
- the rate of dissociation of ligand - receptor complex. Thus if dissociation is faster than separation then you may get bound unbinding then being lost
The speed of separating bound and unbound drugs need to be compatiable with affinity of drug to ligand.
Therefore the lower the affinity the drug is to the receptor, the faster and more efficient separation must be.
How does affinity related to KD?
The relationship is inversely proportional
Thus a lower affinity requires a higher KD
Two things radioligands should be
- binding should be sterospecific
- the radioligand must also be saturable
What does a Scatchard plot tell you?
- The relationship between specifically bound radioactive ligand over the concentration of free non bound ligands.
- the free radioligand is equivalent to the concentration added initially.
On a scatchard plot test, what is on the y and x axis? What does the gradient of the slope tell you?
Y axis is the bound radioligand / free radioligand
The X axis tells you the specific bound concentration
The slope tells you -1/KD
Note a lower KD means a high affinity.
B max is the maxiumum amount of drug which can bind specifically to the receptor in a membrane prep.
