affinity and concentration response

Question 1

the law of mass action shows the binding and dissociation of a drug from its receptor

1. state the equation
2. What constants are represented by K_on and K_off

Answer 1

K_on = association rate constant

K_off = dissociation rate constant

Question 2

true or false?

at equilibrium KD = k_off / k_on

Answer 2

TRUE

Question 3

what are the two main methods for measuring Kd and what do they involve

Answer 3

Radioligand binding

• Direct measurement of drug-receptor interactions using a radiolabelled drug probe

Functional analysis

• Using concentration-response curves

Question 4

what is the equation for receptor occupancy

Answer 4

B = amount bound

B(max) = max amount that can be bound

[D] = concentration of drug

Kd = equilibrium dissociation rate constant

Question 5

define Kd

Answer 5

• K_D - is the (equilibrium) dissociation constant

“The concentration of free drug that occupies 50% of the target population (receptors) at equilibrium”

Question 6

what’s the difference between specific and non-specific binding

Answer 6

1. Drug bound to the receptor - SPECIFIC binding
2. Drug bound to other binding sites - NON-SPECIFIC binding (filters, membranes, other proteins)

Question 7

what is the difference between saturation analysis and competition analysis

Answer 7

• saturation analysis is where the concentration of the radioligand is varied, and the amount of radioligand bound to the receptor is measured
• competition analysis involves keeping the radioligand concentration constant but varying the unlabelled competitor ligand concentration and measuring the amount of radioligand bound to the receptor

Question 8

True or false

the Ki is the same as the Kd

Answer 8

TRUE

Ki is also the dissociation constant

Question 9

what does the IC50 represent?

Answer 9

• The IC₅₀ represents the concentration of the competing ligand that displaces 50% of the specific binding radioligand

Question 10

state the Cheng-Prusoff equation

involving:

Ki

[radioligand]

IC50

K_D(RL)

Answer 10

Question 11

True or False

the lower the Kd/Ki value the lower the affinity

Answer 11

FALSE

• The lower the K_i (KD) value the higher the affinity

Question 12

what drug out of atropine and tiotropium has a higher affinity for thee M3 receptor

Answer 12

• The lower the K_i (KD) value the higher the affinity so we can conclude that tiotropium is 10 fold higher affinity at the M3 receptor than atropine is

Question 13

what does Koff tell us that Kd doesn’t?

and why is this useful?

what does a smaller Koff rate mean?

Answer 13

K_off shows the proportion of the bound drug that dissociated from the target in unit time, whereas Kd just shows the amount of free drug that occupies 50% of the population at equilibrium

this is useful because it allows us to see which drug will have the longest-lasting effects, something Kd cannot show

a smaller K_off rate shows the drug has longer effect at the receptor

Question 14

what two measurements can we get from a concentration-response curve

Answer 14

1. Rmax - the max response
2. EC50 - the effective concentration of the agonist for 50% response

Question 15

define EC50

Answer 15

the effective concentration of the agonist to produce 50% response

Question 16

define a full agonist

Answer 16

• A full agonist is one that has the same maximum response as the reference agonist (in this case adrenaline)

Question 17

define partial agonist

Answer 17

a partial agonist is one that has less efficacy than the reference agonist and therefore has a lower Rmax however it still activates the receptor in the same way

Question 18

explain the concept of receptor reserve

Answer 18

• receptor reserve involves the amplification of a signal by high efficacy agonists to produce a maximal response by only activating a few other receptors, ‘reserve’ refers to the receptors that were not needed to produce this response

Question 19

which image shows a high efficacy agonist exhibiting receptor reserve and which one shows a low efficacy agonist which isn’t

Answer 19

top picture is a low efficacy agonist

bottom picture shows receptor reserve caused by a high efficacy agonist

Question 20

what factors do the potency (EC50) and maximum response (Rmax) of an agonist depend on?

Answer 20

1. Drug-receptor interaction: affinity and efficacy
2. Properties of the functional response i.e. amplification

Question 21

what is 10⁶ = 1000000 in log form

Answer 21

log₁₀1000000 = 6