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Mechanisms of Drug Action > Radioligand Binding > Flashcards

Radioligand Binding Flashcards

1
Q

Does radioligand binding measure response?

A

No

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2
Q

What can iodine be used to tag in radioligand binding?

A

Certain amino acids on polypeptides/proteins

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3
Q

Why are radioligand binding experiments completed?

A
  • Shows how quickly a drug binds and dissociates from the receptor
  • Shows whether a receptor is present in a tissue
  • Shows how many receptors are present
  • Can be used to tell how well unlabelled drug binds to the receptor
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4
Q

Outline how a basic binding assay is completed

A
  • Buffer, radioactive ligand, and cell membranes are incubated
  • Pour incubation over a filter so only free drug molecules pass through
  • Scintillation counter counts reactivity left on filter- producing a value for the drug which has bound to receptors
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5
Q

What are the 3 main types of ligand binding assay?

A
  • Saturation
  • Kinetics
  • Competition/displacement
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6
Q

How is a saturation ligand binding assay completed?

A
  • Run a basic binding assay
  • Repeat experiment with increasing concentrations of radioligand to find total binding
  • Repeat experiment but add the same high concentration of non-radioactive ligand (to find non-specific binding)
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7
Q

Is non-specific binding saturable?

A

No

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8
Q

What is non-specific binding?

A

Binding of a ligand to anything other than a receptor e.g. binding to the membrane

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9
Q

What shape is a non-specific binding-[radioligand] graph?

A

Linear

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10
Q

What shape is a specific binding- [radioligand] graph?

A

Rectangular hyperbola

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11
Q

What does the KD of a binding-concentration graph represent?

A

The concentration of radioligand at which 50% of receptor binding sites are occupied

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12
Q

What condition is required for the values of Bmax and KD to be valid?

A

Equilibrium must have been reached

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13
Q

Briefly evaluate saturation binding

A
  • Expensive as it uses lots of radioligand
  • The only option if only one ligand exists for a receptor
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14
Q

What kind of graph is produced in a kinetics binding asssay?

A

Binding-time graph

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15
Q

Is the plateau from a binding-time graph Bmax?

A

No, it simply shows the maximum binding of that particular concentration of radioligand

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16
Q

Outline how to complete an on-rate kinetics binding assay

A
  • A single concentration of radioligand is incubated for progressively longer times, then filtered and counted (gives total binding)
  • The same experiments are repeated but with addition of a high concentration of competing non-radioactive ligand (gives non-specific binding)
17
Q

Outline how to find the total binding of an off-rate kinetic binding assay

A
  • Set up incubation and leave until eqm has been reached
  • Then disturb eqm by adding a very high concentration of displacing ligand in a large volume of buffer
  • Radioligand dissociates and does not reassociate
  • This is repeated and incubated for different amounts of time
18
Q

Outline how to find the non-specific binding of a kinetic binding assay

A
  • Same as specific binding but with the addition of a large concentration of competing non-radioactive ligand
19
Q

Why are kinetic binding assays completed?

A
  • To determine Kon, Koff, and KD
  • To work out how long it takes for a new ligand to reach eqm
20
Q

Why is Kon hard to determine?

A

Both association and disassociation can occur

21
Q

Outline how to complete a competition binding assay

A
  • Use the same [radioligand] in all tubes but increasing concentrations of displacing non-radioactive ligand, filter and count (total binding)
  • To find non-specific binding use the same [radioligand] with a very high concentration of non-radioactive competing ligand
22
Q

What is IC50?

A

The [displacer] that displaces 50% of a specific radioligand binding

23
Q

What is Ki?

A

The KD of the displacing ligand

24
Q

Is IC50 a constant?

A

No

25
Q

What does IC50 depend upon?

A

The concentration of radioligand used

26
Q

What does a competition binding assay allow you to compare between different ligands?

A

Affinity

27
Q

Can Bmax be found from a competition binding assay?

A

No

28
Q

Why is maximum binding in a competition binding assay less than Bmax?

A

[Radioligand] used is usually around KD to give a decent amount of binding but is not too expensive

29
Q

What are the advantages of competition binding assays?

A
  • Relatively cheap
  • Can analyse a number of ligands at the same time
30
Q

Which type(s) of radioligand binding study is an equilibrium study?

A
  • Saturation
  • Competition
31
Q

Is a kinetic binding assay an equilibrium study?

A

No, it is a non-equilibirum study

32
Q

What is orthosteric binding?

A

Binding to the same site on receptor as endogenous ligand

33
Q

What is allosteric binding?

A

Binding on a site of a receptor which is different to an endogenous ligand

34
Q

Does a receptor only bind to one drug molecule?

A
  • Yes if it is a GPCR
  • Unlikely if it is a ligand gated ion channel receptor
35
Q

What can binding to tissue sections show?

A

The distribution of receptors in tissue types

Mechanisms of Drug Action (8 decks)

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