Theme 2 Lecture 7 Flashcards
How do drugs bind to receptors?
Ligand-receptor interactions are highly specific, binding to distinct amino acid residues within receptor structures.
Understanding these interactions for designing effective drugs and insights into receptor function.
Describe Kd dissociation constant
Kd is when 50% of the receptors are occupied, a low Kd indicates a high affinity.
Defines the concentration of ligand required to bind to 50% of available sites at equilibrium.
What is the law of mass action?
When equilibrium is reached and when the rate of formation of the new receptor-ligand complexes is equal to the rate of dissociation.
How to calculate Kd
Hill-langmuir equation= Fraction of receptors bound
What is the role of fluorophores in ligand labeling?
Enables fluorescent detection of the ligands.
What is a popular fluorophore used in ligand labeling and what is a notable effect of its use?
Bodipy is a popular fluorophore that increases the molecular size of ligands for labelling.
How is ligand binding visualized using fluorophore-tagged ligands?
Microscopy, by exciting the fluorophore with the laser
Name two fluorescent techniques and why they are important?
BRET and FRET, it is essential for studying ligand/receptor interactions.
What is Bmax
The maximum level of specific binding indicates the Bmax, represents the max number of receptors in the tissue.
Measuring Kd and Bmax
High affinity ligands causes the issue.
When the graph is turned into a sigmoidal shape, this makes it easier for the Kd to be read.
The Bmax value should remain the same as the last graph
Why is Kd and Bmax important?
The KD and Bmax values are crucial for understanding ligand binding and receptor characteristics in a tissue.
Non-specific binding
In ligand-binding experiments it is important to be able to distinguish between NSB and SB.
How they isolate it?
1st experiment=Use the ligand to bind to the target receptor which is random.
2nd experiment=Use high concentration of non-radioactive or fluorescent ligands to bind to the specific sites. (saturated)
So only specific binding is prevented and NSB is observed.
Then the difference between both experiments gives the binding curves.
Selecting NSB
The NSB should bind to the same sites and have a high affinity
NSB Graph
Subtract the Top curve from the bottom which gives the green curve, and this isolates the specific binding component.
Measuring association and dissociation rates
It shows us how the substance attaches to and detaches from the receptor as time passes, eventually reaching a balance (equilibrium) where the rates of attaching and detaching are equal.
K off and residence time
“Off rate” is how quickly a drug leaves its target on the cell, and “residence time” is how long the drug stays attached to its target. Longer residence time can mean longer-lasting effects of the drug.
How to calculate K off and residence time?
K off means of 0.01 per minute= 1% of the ligand dissociates from the receptor every minute.
Residence time is the reciprocal of the off rate constant.
If Koff is 0.01, reciprocal means to divide there it would be 1/0.01=100
Measuring the dissociation rate constant
The curve will show a hyperbolic shape (hill)
You can measure dissociation half life which is the time it take for the ligand to binding to reduce by half.
What is the equation used to calculate the dissociation half life?
koff = 0.693 / t1/2
Positive and negative allosteric regulators
Positive= increases residence time for ligands
negative= decreases residence time by increasing dissociation
On rate constant, why is it difficult?
Measuring an on rate constant appears to be more difficult because it involves the rate at which a ligand achieves equilibrium.
An on-rate of 1x10^8 M-1 min-1 means what?
That 1x10^8 will bind per minute per molar free ligand concentration.
equation for k on
Kon = (Kobs - Koff) / Ligand Concentration.
Equation for Kd radioligand
Kd=Koff/Kon
Cheng-Prusoff equation
competitor
Ki= 1+ IC50/1+L/Kd
Ki is the concentration of competitor required to bind 50% receptors sites)
Competition binding equation BR, LR etc
As you increase the concentration of unlabelled competitive ligands it decreases the specific binding of the labelled ligand thus confirming that competitive nature
