Lecture 3 (Receptor Theory I Drug Affinity)

Question 1

What is occupation of receptor by drug molecule governed by?

Answer 1

Affinity (independant of activation)
-Governs number of receptors activated

Question 2

What is activation of receptor by drug molecule governed by?

Answer 2

Efficacy

Question 3

What is affinity regulated by?

Answer 3

Constants (etc.. K+1,K-1)

Question 4

What is the efficacy during an antagonist reaction

Answer 4

Zero

Question 5

What is Kd (dissociation constant)

Answer 5

KD is a constant that defines the affinity of a drug for a receptor

Question 6

What are units of Kd in?

Answer 6

Molarity

Question 7

How do you find Kd? (without graph)

Answer 7

Divide reverse reaction by forward reaction
KD= k-1 / k+1

Question 8

How does high affinity affect Kd number?

Answer 8

A high affinity reaction Kd tends to be very small number (inversely proportional)

Question 9

What is occupancy?

Answer 9

Proportion of receptors occupied will vary with the drug concentration
(varies between 0 and 1)

Question 10

How do you find occupancy?

Answer 10

Occupancy = number of receptors occupied/
total number of receptors

Question 11

How can we use occupancy (at equilibrium) to measure affinity?

Answer 11

Use Radio ligand Binding Assays

Question 12

What us the purpose of tissue in measuring affinity using radioligand binding assays?

Answer 12

Selected to contain the recognition sites (receptors) of interest

Question 13

What conditions are need to preserve integrity of ligand(s) and binding site in method for measuring affinity? (2)

Answer 13

-Keep temp low as enzymes don’t work well at low temp, stops degradation

-Need to add antioxidant as us may have oxidable molecules

Question 14

What are the conditions for the radioligand in needed in measuring affinity using radioligand binding assays? (3)

Answer 14

-Radioactive drug must be very pure

-Degradation must be avoided

-Must have very high specific activity to allow very low (“tracer”) concentrations.

Question 15

How can you avoid degradation of radioligand? (4)

Answer 15

1. free-radical scavenger (eg ethanol) in drug solution.
2. store at low (not freezing) temperature.
3. Avoiding light (dark bottles to store).
4. incorporation of antioxidant (eg. ascorbic acid).

Question 16

How is separating tissue+bound ligand from free ligand remaining in incubation media is usually done? what other techniques are used?

Answer 16

Filtration or centrifugation

Dialysis, column chromatography, precipitation/adsorption

Question 17

What must the speed of separation of tissue+bound ligand from free ligand be compatible with?
What conditions are required for fast separation?

Answer 17

-Affinity of ligand for receptor

-Low affinity

Question 18

What is Non-specific binding in test for drug affinity?

Answer 18

Ligand in addition to binding to receptor may bind to other things in test tube such as the plastic it is made up of, or other proteins found in preparation

Question 19

What is the solution to Non-specific binding in test for drug affinity? (2) How do you take it away?

Answer 19

-Done by setting up 2 test tubes in parallel with the same receptors and tissues in it
-In one test tube add known conc of radiative drug and the same to the other one but you add a huge excess of non-radioactive drug into one of them

-Non-radioactive drug out competes radioactive drug for specific binding as it is higher affinity
-Only radioactivity bound in nonspecific
(Take away non-specific binding from total)

Question 20

What is the equation for specific binding?

Answer 20

Specific binding = total bound – non specific binding

Question 21

What kind of scale is used for Radioligand binding curves?

Answer 21

semi-logarithmic

Question 22

What does it mean when graphs plateaus on radioligand binding curve?

Answer 22

When it plateaus when you know all receptors are bound to ligands

Question 23

What does higher affinity for drug mean? (in terms of occupancy)

Answer 23

The higher the affinity of a drug for its receptor, the lower the concentration will be needed for a given level of occupancy

Question 24

What does the concentration of drug at which 50% receptors are OCCUPIED by DRUG provide?

Answer 24

Measure of affinity (Kd)

Question 25

What effect is there when two drugs compete for same receptor?

Answer 25

Reduces apparent affinity for other drug

Question 26

How can we compare receptors in different tissues?

Answer 26

Can compare binding of drug between two tissues to see if same receptors are there