ELM3: Drug binding to receptors

Question 1

What is the law of mass action?

Answer 1

Mathematical framework for rate of reaction
Rate of reaction is directly proportional to concentrations of reactants

Question 2

What are the two background principles for the Hill-Langmuir equation?

Answer 2

Drug binding is saturable
Drugs have affinity for their targets

Question 3

What is meant by the principle of drug binding being saturable?

Answer 3

Finite number of drug binding sites
The max number of drug molecules that can bind to cell is Bmax

Question 4

What is meant by the principle of drugs having affinity for their targets?

Answer 4

Affinity is attraction
Measured with KD

Question 5

What is KD?

Answer 5

Equilibrium dissociation constant governing reaction of drug and receptor binding

Question 6

What is the relationship between KD and affinity?

Answer 6

Inversely proportional

Question 7

What is the shape yielded by a plot of B vs [D]?

Answer 7

Rectangular hyperbola

Question 8

What occurs when [D] is higher than KD?

Answer 8

Value of B is close to Bmax

Question 9

How is Bmax determined using the graph?

Answer 9

Where the plateau at the top of the curve is

Question 10

What occurs when [D] is the same as KD?

Answer 10

B = Bmax

Question 11

How can we work out KD?

Answer 11

1. Find Bmax
2. Draw a line across Bmax/2
3. Find where intersects curve and follow down to x axis

Question 12

What is a radioligand binding assay?

Answer 12

Quantifies how much of a drug has bound in a tissue sample
Allows us to detect the binding using a radiolabelled version of the drug

Question 13

What is the name of radiolabelled drugs?

Answer 13

Radioligands

Question 14

What are three common radiolabels?

Answer 14

3H 14C 125I

Question 15

What is the overall three steps of radioligand binding assays?

Answer 15

Incubate
Separate
Quantify

Question 16

What is the incubate step of radioligand binding assays?

Answer 16

1. Incubate ligand and tissue until equilibrium

Question 17

What is the separate step of radioligand binding assays?

Answer 17

Uses filtration or centrifugation
1. Radioligand assays made by mixing receptors and radioligand
2. Come to equilibrium
3. Some radioligand bind to receptors and some don’t
4. In filtration assay is passed through glass fibre with 1um pores
5. Free radioligand is small and passes through

Question 18

What is the quantify of radioligand binding assay?

Answer 18

1. Measure amount of radioactivity in a sample

Question 19

What method is used to quantify beta emitting nucleotides like 3H?

Answer 19

Liquid scintillation counting

Question 20

What method is used to quantify gamma emitters like 125I?

Answer 20

Gamma counter

Question 21

What is non-specific binding?

Answer 21

When a radioligand binds but not to the actual receptor and instead another part of the cell

Question 22

How is a control sample prepared?

Answer 22

Saturation assay
Non radioactive ligand is added which will bind to all of the receptor sites
This allows the radioligand to bind to the non specific binding sites
Allows us to count non specific binding

Question 23

What can a saturation assay tell us about a tissue of interest?

Answer 23

Affinity (KD) and receptor density (Bmax)

Question 24

What are two limitations of doing a saturation assay?

Answer 24

Needs a radioactive version of every drug being tested which is expensive
Needs a high concentration of radioligand which is expensive and makes radioactive waste

Question 25

How can we compare the affinities of different drugs?

Answer 25

Use saturation assay to get KD value and compare affinity with another drug
KD and affinity are inversely proportional

Question 26

How can we compare the selectivity of two drugs?

Answer 26

Use KD to compare the drugs affinities at two different receptors

Question 27

What is the definition of an enzyme?

Answer 27

Biological catalysts that speed up the transformation of a substrate into a product

Question 28

What are the two steps of the reaction between an enzyme and substrate?

Answer 28

Binding step that forms complex
Catalytic step that generates product

Question 29

How is the Hill-Langmuir equation modified to fit enzyme activity

Answer 29

Michaelis Menton
Binding step of enzymes is equivalent to a drug and a receptor