Receptor theory II Flashcards
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?
Activation
The change in the cell, by causing the stabilisation of the active agonist/receptor configuration
What does efficacy apply to?
Agonists only
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?
0-1
1 = all receptors occupied
0 =no receptors occupied
What is occupancy NOT proportional to?
Why?
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:
- Affinity
- 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
Measure affinity
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
Then:
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)
- Degradation
- 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?
Ethanol
Example of an antioxidant?
Asorbic acid
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
Problems:
- 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
What is the shape of the non-specific binding curve?
Linear
What is the shape of the specific binding curve?
What does this show?
Increase rapidly to a peak
Then level off as reach a maximum
Shows saturation
How are specific binding graphs plotted and why?
X axis on a semi-log scale
Fit more concentrations on a reasonably sized graph
What does the Langmuir equation describe?
What is it a plot of?
Describes the relationship between drug concentration and occupancy (specific binding –> therefore affinity)
On the X axis - concentration of drug (on a log scale)
On the Y axis - Specific binding
How do you work out the Kd and Bmax from the Langmuir plot?
Bmax is the the level at which the graph plateaus
Kd is the measure at 50% occupancy of the total number of receptors
What 3 things is the amount of bound ligand dependant on?
1) Bmax
2) Concentration of the drug
3) Kd (Binding coefficient)
How is the Kd related to the same receptor in different tissue?
It is the same value if it is the same receptor-ligand match but in different tissues
As Kd is a measure of affinity
What does Kd detetmine
Affinity
The on/off rate of the ligand to the receptor
What is the scatchard equation?
B/F =(Bmax-B)/KD
B = specifically bound radioligand F = free radio ligand ( ~ concentration added) Bmax = maximum no. of binding sites KD = equilibrium binding constant (affinity)
OR
B/F = - 1/Kd (B- Bmax)
What is the scatchard plot and why is it useful?
Rearragement of the Langmuir equation - to make a linear plot
Better to calculate Kd
Describe to scatchard plot
On the X axis:
- Specific bound radioligand
- X intercept is the Bmax
On the Y axis:
- B/F (bound over free radioligand)
Downwards linear plot where the slope is -1/KD
How to calculate the Kd from the slope in the scatchard plot?
Slope = Y/X
Slope = -1/Kd
Kd= -1/slope
Why is the ‘free concentration of drug’ the same as the concentration of drug added in the first place?
No. of drug molecules bound to receptors is insignificant
What is the Bmax and how is this useful?
The maximum amount of receptors in the membrane which the drug can bind to
- Can compare the Bmax in health and disease (is there a decrease in receptors?)
What is the relationship between Kd and affinity?
Inverse relationship
Low Kd - high affinity (don’t need much of the drug)
High Kd - low affinity (need lots fo the drug to cause a response)
What is the shape of a langmuir plot?
Sigmoidal
How can you work out the Kd from a langmuir plot?
50% occupancy
What can you work out from the scatchard equation?
Kd (and therefore the affinity of the agonist)
Bmax
