Drug Binding

Question 1

What is the Law of Mass Action?

Answer 1

Describes the relationship between reactants and products in reversible reactions.

It states that the rate of a chemical reaction is proportional to the product of the concentrations of the reactants.

Question 2

What is the equation representing the reaction of oxygen and wood?

Answer 2

Oxygen + wood -> carbon dioxide + water.

This represents a combustion reaction.

Question 3

What does the Hill-Langmuir Equation describe?

Answer 3

The relationship between drug concentration and the binding of drugs to receptors.

It accounts for saturable binding sites in cells and tissues.

Question 4

Define B_max in the context of drug binding.

Answer 4

The maximum number of binding sites available for a drug. This is relative to the amount of protein in a sample

B_max is measured in fmol/mg.

Question 5

What does K_D represent?

Answer 5

Equilibrium dissociation constant indicating the affinity of a drug for its target.

It is defined as K_D = 1/K_eq.

Question 6

What happens to drug affinity as K_D decreases?

Answer 6

Affinity increases as K_D goes down.

Lower K_D values indicate stronger binding of the drug to the receptor.

Question 7

What is the significance of the concentration of drug that occupies half of the binding sites?

Answer 7

It is equal to K_D.

This reflects the potency of the drug.

Question 8

Fill in the blank: When [D] is much greater than K_D, the binding is _______.

Answer 8

saturated.

The value of B is close to Bmax

Question 9

What does the Michaelis-Menten Equation relate to?

Answer 9

The kinetics of enzyme-catalyzed reactions.

It describes the relationship between substrate concentration and reaction rate.

Question 10

What does K_m represent in the Michaelis-Menten Equation?

Answer 10

The concentration of substrate at which the reaction rate is half of V_max.

It is analogous to K_D in drug binding.

Question 11

What is selectivity in pharmacology?

Answer 11

The comparison of a drug’s affinity for different receptor types.

It is crucial for reducing side effects in drug development.

Question 12

What are the advantages of radioligand binding assays?

Answer 12

• Simple
• Cheap
• Flexible
• Scalable
• Sensitive

These assays allow for the measurement of drug binding to receptors.

Question 13

What are the disadvantages of radioligand binding assays?

Answer 13

• Hazardous to work with
• Waste difficult to dispose of

Handling radioactive materials poses safety and regulatory challenges.

Question 14

What is a radioligand?

Answer 14

A version of a drug with a radioactive label added.
Added to drugs to detect binding and quantify how much drug has bound in a tissue sample

Common isotopes used include 3H (tritium), 14C, or 125I.

Question 15

How is specific binding calculated in radioligand assays?

Answer 15

Specific binding = total binding - non-specific binding (NSB).

NSB refers to the binding to low-affinity sites.

Question 16

True or False: Non-specific binding sites are saturable.

Answer 16

False.

Non-specific binding sites represent a vast pool of low-affinity interactions.

Question 17

What is a saturation assay used for?

Answer 17

To find K_D and B_max for a radioligand.

It helps determine how tightly a drug binds to its receptor.

Question 18

What are the problems associated with saturation assays?

Answer 18

• Need a radioactive version of every drug you want to investigate
• Expensive to make
• Time-consuming to make
• Produces lots of radioactive waste
• Aren’t cheap
• Need to use high concentrations
• Impractical for drug screening

Question 19

What is the role of competition assays as an alternative?

Answer 19

To assess drug binding without the need for radioactive versions of every drug.

They utilize competitive inhibition to analyze binding properties.

Question 20

What is the importance of analyzing enzyme assay data?

Answer 20

It helps in understanding the kinetics of enzyme reactions and drug interactions.

Accurate data analysis is crucial for drug development.

Question 21

Fill in the blank: Curve fitting vs _______ is a method of analyzing enzyme kinetics.

Answer 21

linear transformation.

Question 22

What is the Hill-Langmuir equation?

Answer 22

B = Bmax X [D]
——————
KD + [D]

Where
Bmax = maximal binding capacity
B = concentration of drug-receptor complex (DR)
KD = equilibrium constant for drug at the receptor
[D] = concentration of the drug

Question 23

What does the typical plot for a Hill-Langmuir equation look like?

Answer 23

Binding on the Y axis and [Drug] on the X axis

Rectangular hyperbola shape

The asymptotes of the curve meet at right angles

Question 24

How to find Bmax?

Answer 24

Estimate where the plateau is and follow it to the Y axis

Question 25

What happens when the concentration of D is equal to KD?

Answer 25

[B] is equal to half of Bmax

Question 26

How to find value of KD?

Answer 26

KD is the concentration of drug that occupies half the binding sites Find half of Bmax on the Y axis and follow it down to the X axis

Question 27

What is the Michaelis-Menten Equation?

Answer 27

V = Vmax X [S] —————— Km + [S] Where V = initial velocity of the reaction (a measure of the concentration of the enzyme-substrate complex) Vmax = maximum rate (max E-S complexes) Km = equivalent to KD

Question 28

What does a low Km indicate?

Answer 28

Tighter binding

Question 29

What is Vmax directly proportional to?

Answer 29

Enzyme concentration

Question 30

What are the 3 steps in radioligand binding assays?

Answer 30

1. Incubation = where binding takes place 2. Separation = removal of any excess unbound radioligand 3. Quantification = measurement of how much has bound

Question 31

Describe incubation & separation in binding assays

Answer 31

Initiated by mixing together the radioligand and receptors 1. Receptors are embedded in the membrane and the radioligand is in solution around the membrane 2. Radioligand attaches to receptors (specific binding) and some bind to sites on the membrane (non-specific binding). Most of the radioligand remains unbound 3. Assay mixture is pushed through a fine 1 um filter where membranes are retained 4. Unbound ligand passes through the filter 5. After washing to remove any residual unbound radioligand, the amount of radioactivity on the filters can be measured

Question 32

Describe quantification in binding assays

Answer 32

Can be done via - Liquid scintillation counting (LSC) —> uses beta emitting nuclides such as [3H] = adding a solution containing components that will emit light when struck by radiation so can measure the amount of light produced - Gamma counter using [125I] —> tube with sample is placed next to a solid scintillation and gamma rays strike the solid scintillation which then emits light = intensity of light is a measure of radioactivity in the sample

Question 33

Quantifying non-specific binding (NSB)

Answer 33

- Extremely large pool of low affinity sites - Specific binding to the receptor = tiny pool of high affinity sites - Measure total binding - Specific binding = total binding - NSB - Measure binding in presence of high concentration of non-radioactive drug

Question 34

Describe NSB binding drugs

Answer 34

Used in large concentrations Binds and occupies all receptor sites = radioligand only binds to sites on the membrane

Question 35

Equipment and materials needed to perform a saturation assay

Answer 35

- Tissue sample containing receptor - Radioligand that binds to receptors tightly (must be over 10 nm) - Non-radioactive drug that binds to the same sites as the radioligand - Filtration system to separate the bound from the free - Radiation counter to detect bound ligand

Question 36

How do you manually plot an analysis of saturation data?

Answer 36

1. Plot data on a graph where the Y axis is binding and the X axis is the concentration of the drug 2. Draw a smooth curve through the data = rectangular hyperbola shape 3. Estimate the Bmax value where the curve plateaus 4. Find the value of half Bmax 5. Find the KD value by following half Bmax to the X axis = intersection with the X axis