Drug Binding Flashcards
What is the Law of Mass Action?
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.
What is the equation representing the reaction of oxygen and wood?
Oxygen + wood -> carbon dioxide + water.
This represents a combustion reaction.
What does the Hill-Langmuir Equation describe?
The relationship between drug concentration and the binding of drugs to receptors.
It accounts for saturable binding sites in cells and tissues.
Define B_max in the context of drug binding.
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.
What does K_D represent?
Equilibrium dissociation constant indicating the affinity of a drug for its target.
It is defined as K_D = 1/K_eq.
What happens to drug affinity as K_D decreases?
Affinity increases as K_D goes down.
Lower K_D values indicate stronger binding of the drug to the receptor.
What is the significance of the concentration of drug that occupies half of the binding sites?
It is equal to K_D.
This reflects the potency of the drug.
Fill in the blank: When [D] is much greater than K_D, the binding is _______.
saturated.
The value of B is close to Bmax
What does the Michaelis-Menten Equation relate to?
The kinetics of enzyme-catalyzed reactions.
It describes the relationship between substrate concentration and reaction rate.
What does K_m represent in the Michaelis-Menten Equation?
The concentration of substrate at which the reaction rate is half of V_max.
It is analogous to K_D in drug binding.
What is selectivity in pharmacology?
The comparison of a drug’s affinity for different receptor types.
It is crucial for reducing side effects in drug development.
What are the advantages of radioligand binding assays?
- Simple
- Cheap
- Flexible
- Scalable
- Sensitive
These assays allow for the measurement of drug binding to receptors.
What are the disadvantages of radioligand binding assays?
- Hazardous to work with
- Waste difficult to dispose of
Handling radioactive materials poses safety and regulatory challenges.
What is a radioligand?
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.
How is specific binding calculated in radioligand assays?
Specific binding = total binding - non-specific binding (NSB).
NSB refers to the binding to low-affinity sites.
True or False: Non-specific binding sites are saturable.
False.
Non-specific binding sites represent a vast pool of low-affinity interactions.
What is a saturation assay used for?
To find K_D and B_max for a radioligand.
It helps determine how tightly a drug binds to its receptor.
What are the problems associated with saturation assays?
- 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
What is the role of competition assays as an alternative?
To assess drug binding without the need for radioactive versions of every drug.
They utilize competitive inhibition to analyze binding properties.
What is the importance of analyzing enzyme assay data?
It helps in understanding the kinetics of enzyme reactions and drug interactions.
Accurate data analysis is crucial for drug development.
Fill in the blank: Curve fitting vs _______ is a method of analyzing enzyme kinetics.
linear transformation.
What is the Hill-Langmuir equation?
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
What does the typical plot for a Hill-Langmuir equation look like?
Binding on the Y axis and [Drug] on the X axis
Rectangular hyperbola shape
The asymptotes of the curve meet at right angles
How to find Bmax?
Estimate where the plateau is and follow it to the Y axis
What happens when the concentration of D is equal to KD?
[B] is equal to half of Bmax
How to find value of KD?
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
What is the Michaelis-Menten Equation?
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
What does a low Km indicate?
Tighter binding
What is Vmax directly proportional to?
Enzyme concentration
What are the 3 steps in radioligand binding assays?
- Incubation = where binding takes place
- Separation = removal of any excess unbound radioligand
- Quantification = measurement of how much has bound
Describe incubation & separation in binding assays
Initiated by mixing together the radioligand and receptors
- Receptors are embedded in the membrane and the radioligand is in solution around the membrane
- Radioligand attaches to receptors (specific binding) and some bind to sites on the membrane (non-specific binding). Most of the radioligand remains unbound
- Assay mixture is pushed through a fine 1 um filter where membranes are retained
- Unbound ligand passes through the filter
- After washing to remove any residual unbound radioligand, the amount of radioactivity on the filters can be measured
Describe quantification in binding assays
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
Quantifying non-specific binding (NSB)
- 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
Describe NSB binding drugs
Used in large concentrations
Binds and occupies all receptor sites = radioligand only binds to sites on the membrane
Equipment and materials needed to perform a saturation assay
- 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
How do you manually plot an analysis of saturation data?
- Plot data on a graph where the Y axis is binding and the X axis is the concentration of the drug
- Draw a smooth curve through the data = rectangular hyperbola shape
- Estimate the Bmax value where the curve plateaus
- Find the value of half Bmax
- Find the KD value by following half Bmax to the X axis = intersection with the X axis
