Measuring Receptor Binding/Signalling

Question 1

Kd is defined as:

Answer 1

The concentration of drug that binds FO=0.5, when

Kd= (L)

Question 2

What is Bmax?

Answer 2

The number of receptors in the preparation (FO=1)

Question 3

What may the objectives of Direct measurement of drug binding to receptors?

Answer 3

• Determine the receptor binding properties of the ligand (Kd)
• Determine properties of the receptor.

Question 4

What might you do to determine the properties of the ligand?

Answer 4

• Screen a receptor against a compound of interest
• Understand the association and dissociation rates of the ligand (affinity) (is dissociation so slow its nearly irreversible?)

Question 5

What properties of a receptor can be determined?

Answer 5

-Receptor density of a cell (Bmax)

_ How diseases or drugs change the Bmax or ability of a receptor to bind to drugs

Question 6

What is a common feature of all radioligand assays?

Answer 6

All radioligand assays:

• Require you to have a radioactively labelled,
  1) High affinity
  2) Highly selective
  3) High specific activity compound
• Require you to incubate radiolabelled compound with tissue of interest and then separate bound drug from free.

Question 7

What are three properties of a radioligand?

Answer 7

1) High affinity
2) Highly selective
3) High specific activity compound (n. radioactive molecules attached to compound?)

Question 8

what are the sorts of tissues of interest?

Answer 8

• Whole cells adhered to culture plates
• Crude homogenised tissue (blended tissues, no idea which receptors belonged to what)
• Enriched cell membrane preparations
• Tissue adhered to microscope slides

Question 9

How do you seperate bound from free?

Answer 9

A setup consisting of:
Filter Plate
Glass fiber filter paper- proteins bind to this.
wash off non-bound radioligand and all that is left is cells with radioligand bound to it.

Question 10

What are the four basic principles of Competition and Saturation binding assays?

Answer 10

1) Carried out under equilibrium conditions (Rate on = Rate off, (must wait till this occurs before non-bound wash off)
2) Incubate samples of tissue with various concentrations of radioactive drug until equilibrium is reached
3) Seperate bound from free
4) Measure bound radioactivity

Question 11

What is competition and saturation assays most commonly performed on?

Answer 11

Most commonly performed on cell membrane homogenates with bound and free separated by filtration

Question 12

What is a saturation binding assays?

Answer 12

Where 100% of receptors are bound to determine receptor density/ Bmax and Kd.

Question 13

How is a saturation binding assay done?

Answer 13

• Incubate membranes with increasing concentrations of radioligand (allow equilibrium to be reached)
• Separate bound from free by filtration through filter
• Dissolve filter in scintillation fluid and count bound radioactivity

Question 14

What is non-specific binding?

Answer 14

When a drug binds to something other than a receptor. i.e membrane

Question 15

Why do compounds have non-specific binding?

Answer 15

Due to:

• Distribution of ligand into lipid components of the preparation
• Free ligand which is not separated from bound ligand during the separation phase of the experiment.

Question 16

What is a feature of non-specific binding that would be expressed on a saturation curve?

Answer 16

They are non-saturable and will increase linearly with radioligand concentration.

Question 17

How can non-specific binding be estimated?

Answer 17

Non-specific binding can be estimated by measuring the binding of the radioligand in the presence of an agent which is believed to bind selectively to the receptor, at a concentration which is calculated to prevent virtually all specific binding without appreciable modification of non-specific binding.

i.e use a ligand that prevent radioligand binding to the receptors, thus any radioligand is non-specific

Question 18

Using saturation binding, on a graph how is specific binding determined?

Answer 18

Total binding - NSB = Specific Binding

Question 19

Do different tissues allow their receptors to have different affinities?

Answer 19

No, Affinity is suppose to be independent of tissue.

Question 20

What are competition assays?

Answer 20

The use of two ligands that bind to the receptor, one which is radioactively labelled, to determine the IC50(concentration of drug that prevents 50% radioligand binding) of the non-labelled ligand.

i.e The relative affinity of a drug (typically new developments (compare it to a known drug that has been labelled)

Question 21

What is the method behind a competition assay?

Answer 21

• A fixed amount of ligand at a concentration below or close to its Kd is equilibriated with the receptor preparation in the presence of a range of increasing concentrations of the unlabelled drug.

Unlabelled drug inhibits labelled binding if they bind to the same receptor.

Must wait till equilibrium is reached, then separate bound from free to measure

Question 22

in competition assays, the radioligand must be at what concentration?

Answer 22

Near to or at its Kd. This concentration is referred to as Bo on a competition curve. (Not the same as Bmax)

Question 23

The steepness of a competition curve should be defined as: or else?

Answer 23

2 log units

otherwise:
Steeper= allosteric binding may be occuring
Shallower= Multiple receptors

Question 24

What is the major conclusion of a comeptition curve?

Answer 24

0.5 Bo (+nsb) = Log IC50

Question 25

IC50 is?

Answer 25

RELATIVE affinity of a compound for a receptor

Question 26

What can IC50 be used for?

Answer 26

• Comparing relative affinity of drugs to compete off radioligand
• To determine the ligands Ki (actualy affinity)

Question 27

How is IC50 used to determine Ki?

Answer 27

KL= Radioactive ligand affinity constant
Need to know the dissociation constant of the inhibitor…

Thus

Ki = IC50 / 2

2= 1 + (ligand concetration) / KL

Question 28

Things to consider;

Answer 28

When would you do a competition assay vs saturation assay?

If you discovered a new ligand how would you determine which receptor it bound to?