Lecture 3 - Drug affinity

Question 1

Definition of a ligand

Answer 1

any molecule which binds to a receptor, agonist or antagonist

Question 2

What is the meaning of drug affinity?

Answer 2

How well a drug binds to a receptor.

Question 3

What is the term KD?

what is the link between KD and affinity?

Answer 3

a constant which describes affinity for a drug to its receptor.

KD is equivalent to conc of a drug required to occupy 50% of receptors, at equilibrium.

Low KD = high affinity
High KD = low affinity.

Question 4

What is the meaning of occupancy?

Answer 4

occupancy is governed by affinity = proportion of receptors occupied that will vary with the drug concentration (varied between 0 and 1, 0 = no drug present, 1 = all receptors occupied)

Question 5

What is the meaning of efficacy?

Answer 5

How well it activates the receptor or evokes a response once bound.

• Antagonists have an efficacy of zero while FULL agonists have an efficacy if 1.
• Some agonists (even when they occupy all receptors) cannot produce a full response, so are partial agonists and have an efficacy between 0 and 1.

Question 6

In methods to measure drug affinity, what 4 factors are important?

Answer 6

1. Tissue and incubation conditons.
2. The radioligand.
3. Seperating bound from free.
4. Non-specific binding

Question 7

How are tissue and incubation conditons important?

Answer 7

Tissue –> selected to contain the recognition sites eceptors) of interest.

Incubation - needs to preserve integrity. Additives” used to protect tissue/ligand of ligands and binding.

• incubation temp is also important.

Question 8

Importance of the radioligand?

Answer 8

• needs to be biologically active
• ligand needs to be extremely pure chemically.
• drug has to be labelled with radioactivity that will allow high specificity so there are very low tracer concentrations.
• Degradation: can be a major problem - solved by..
  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).
• And there are 2 choices of radiolabels H or I.

Question 9

Why is seperating bound from free important in practical considerations of measuring receptor affinity?

Answer 9

tissue and bound ligand is seperated from free ligand remaining in incubation media by filtration or centrifugation.

BUT with soluble binding, other receptors are used: dialysis, column chromatography, precipitation/adsorption.

Question 10

What is a major problem in seperating bound ligand from free?

Answer 10

The major problem is RATE of dissociation of ligand-receptor complex.

Speed of separation must be compatible with affinity of ligand for receptor.

Lower affinity (higher KD) requires faster/more efficient separation.

Question 11

Why is non-ligand binding an important practical consideration?

Answer 11

Anti-absorbants can be used to reduce non-specific binding e.g to filters or glass (eg. albumin or collagen for peptides, o-catechol for catecholamines).

But this does not reduce non-specific binding to the tissue under study. Measuring proportion of specific and non-specific binding is KEY element to assay.
**Rinsing only removes unbound radioligand from incubation medium

Question 12

what is specific binding?

Answer 12

it is the total bound - the non-specific binding.

specific binding shoes saturation (and a sigmoid curve) whilst non-specific does not.