Flashcards in Receptor theory II Deck (47)
What is the binding of an agonist governed by?
The rate of the forwards and backwards reaction of binding
Affinity - balance between the 2
What is affinity and what it a balance of?
A measure of how well the drug binds to its receptor
The balance between the forwards and backwards reaction
(high affinity, high forwards reaction and slow reverse reaction)
What is occupancy a measure of and what is it dependant on
How many receptors are occupied by a drug at any moment in time
Dependant on AFFINITY for the drug
What is an antagonist?
A drug which binds to a receptor, without switching on receptor signalling - doesn't directly
What drugs does affinity apply to?
Equally applies to agonists and antagonists
What is affinity constant for?
A specific drug with its specific receptor in ANY tissue
What does efficacy govern?
The change in the cell, by causing the stabilisation of the active agonist/receptor configuration
What does efficacy apply to?
On what scale is efficacy measures?
On a scale of 0-1
High efficacy is a value of 1
In the case of an agonist, how is affinity related to efficacy?
They are in dependant of each-other
Reactions can have a high affinity and a low efficacy and vice versa
What is the efficacy of an antagonist?
0 - Unable to induce an active configuration
What is occupancy a measure of?
The proportion of receptors occupied with a certain drug concentration
On what scale is occupancy measured?
1 = all receptors occupied
0 =no receptors occupied
What is occupancy NOT proportional to?
The size of the response
Because the maximum response of a tissue can occur with less than 100% occupancy - the tissue can have spare receptors
Maximum response of a tissue can occur at <5% occupancy
Why is measuring the response not a good way to measure occupancy in order to calculate affinity?
Size of the response is governed by both:
- Efficacy in agonists
(not just affinity)
What assay is used to measure occupancy to get an idea of affinity?
Radio ligand binding assay
What does a radio ligand binding assay measure?
Biding of a ligand (agonist or antagonist) to a protein target
What is the main negative point of a radio ligand binding assay?
How is this overcome?
NONE-SPECIFIC BINDING occurs
- Overcome by doing 2 experiments in parallel
- Slowly increase concentration of ligand in each tube
- Had a HUGE excess to one of the tubes
- Displaces the specific binding and adds to the non-specific binding
Tube 1 - Radio active drug is specific and non-specific
Tube 2- Radio active drug is ONLY NON-SPECIFIC
SO tube 1 -tube 2 is specific binding of the radioactive drug
When choosing the radio-ligand, what must be considered?
- Purity (consider isomers)
- How to label drug
What 4 steps can be taken to minimise the degradation of the radioactive drug?
1) Low temperature
2) Add oxidant (minimise oxidation)
3) Avoid light (can cause chemical changes)
4) Add free-radical scavengers
Example of a free-radical scavenger?
Example of an antioxidant?
What are 3 advantages of Tridium as a radioactive substance?
- Doesn't alter properties
- High specific activities
- Long half-life (good stability)
What are 3 disadvantages of Tridium as a radioactive substance?
- Dangerous to humans
- Specialist labs needed (expensive)
- Amount of label per molecule differs
What are the 2 advantages of using radioactive iodine as a radioactive substance?
- Easy and cheap
- Can be incorporated at high specific activities
What are the 2 disadvantages of using radioactive iodine as a radioactive substance?
- Short half-life
- Biological activity of the ligand measured can be reduced
Why must the tissue be incubated?
When blend cells to release receptors - breaks up lysosomes and releases enzymes
- Incubating prevents enzymes from breaking down the proteins and receptors
How separate bound ligand (specific or non-specific) from non bound?
What are the problems with this method?
Centrifugation - Forms a pellet containing the protein
- Specific binding may occur to the pellet (must rinse)
All these steps take time:
- If the drug has a low affinity, off rate is quick
- When washing pellet, may wash off some previously bound ligand
What is Kd and how does it relate to affinity?
Kd - binding coefficient
The higher the Kd, the lower the affinity
Weaker the ligand is bound