Theme 2 Lecture 7

Question 1

How do drugs bind to receptors?

Answer 1

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.

Question 2

Describe Kd dissociation constant

Answer 2

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.

Question 3

What is the law of mass action?

Answer 3

When equilibrium is reached and when the rate of formation of the new receptor-ligand complexes is equal to the rate of dissociation.

Question 4

How to calculate Kd

Answer 4

Hill-langmuir equation= Fraction of receptors bound

Question 5

What is the role of fluorophores in ligand labeling?

Answer 5

Enables fluorescent detection of the ligands.

Question 6

What is a popular fluorophore used in ligand labeling and what is a notable effect of its use?

Answer 6

Bodipy is a popular fluorophore that increases the molecular size of ligands for labelling.

Question 7

How is ligand binding visualized using fluorophore-tagged ligands?

Answer 7

Microscopy, by exciting the fluorophore with the laser

Question 8

Name two fluorescent techniques and why they are important?

Answer 8

BRET and FRET, it is essential for studying ligand/receptor interactions.

Question 9

What is Bmax

Answer 9

The maximum level of specific binding indicates the Bmax, represents the max number of receptors in the tissue.

Question 10

Measuring Kd and Bmax

Answer 10

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

Question 11

Why is Kd and Bmax important?

Answer 11

The KD and Bmax values are crucial for understanding ligand binding and receptor characteristics in a tissue.

Question 12

Non-specific binding

Answer 12

In ligand-binding experiments it is important to be able to distinguish between NSB and SB.

Question 13

How they isolate it?

Answer 13

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.

Question 14

Selecting NSB

Answer 14

The NSB should bind to the same sites and have a high affinity

Question 15

NSB Graph

Answer 15

Subtract the Top curve from the bottom which gives the green curve, and this isolates the specific binding component.

Question 16

Measuring association and dissociation rates

Answer 16

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.

Question 17

K off and residence time

Answer 17

“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.

Question 18

How to calculate K off and residence time?

Answer 18

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

Question 19

Measuring the dissociation rate constant

Answer 19

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.

Question 20

What is the equation used to calculate the dissociation half life?

Answer 20

koff = 0.693 / t1/2

Question 21

Positive and negative allosteric regulators

Answer 21

Positive= increases residence time for ligands
negative= decreases residence time by increasing dissociation

Question 22

On rate constant, why is it difficult?

Answer 22

Measuring an on rate constant appears to be more difficult because it involves the rate at which a ligand achieves equilibrium.

Question 23

An on-rate of 1x10^8 M-1 min-1 means what?

Answer 23

That 1x10^8 will bind per minute per molar free ligand concentration.

Question 24

equation for k on

Answer 24

Kon = (Kobs - Koff) / Ligand Concentration.

Question 25

Equation for Kd radioligand

Answer 25

Kd=Koff/Kon

Question 26

Cheng-Prusoff equation
competitor

Answer 26

Ki= 1+ IC50/1+L/Kd

Ki is the concentration of competitor required to bind 50% receptors sites)

Question 27

Competition binding equation BR, LR etc

Answer 27

As you increase the concentration of unlabelled competitive ligands it decreases the specific binding of the labelled ligand thus confirming that competitive nature