ELM3: Drug binding to receptors Flashcards
What is the law of mass action?
Mathematical framework for rate of reaction
Rate of reaction is directly proportional to concentrations of reactants
What are the two background principles for the Hill-Langmuir equation?
Drug binding is saturable
Drugs have affinity for their targets
What is meant by the principle of drug binding being saturable?
Finite number of drug binding sites
The max number of drug molecules that can bind to cell is Bmax
What is meant by the principle of drugs having affinity for their targets?
Affinity is attraction
Measured with KD
What is KD?
Equilibrium dissociation constant governing reaction of drug and receptor binding
What is the relationship between KD and affinity?
Inversely proportional
What is the shape yielded by a plot of B vs [D]?
Rectangular hyperbola
What occurs when [D] is higher than KD?
Value of B is close to Bmax
How is Bmax determined using the graph?
Where the plateau at the top of the curve is
What occurs when [D] is the same as KD?
B = Bmax
How can we work out KD?
- Find Bmax
- Draw a line across Bmax/2
- Find where intersects curve and follow down to x axis
What is a radioligand binding assay?
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
What is the name of radiolabelled drugs?
Radioligands
What are three common radiolabels?
3H 14C 125I
What is the overall three steps of radioligand binding assays?
Incubate
Separate
Quantify
What is the incubate step of radioligand binding assays?
- Incubate ligand and tissue until equilibrium
What is the separate step of radioligand binding assays?
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
What is the quantify of radioligand binding assay?
- Measure amount of radioactivity in a sample
What method is used to quantify beta emitting nucleotides like 3H?
Liquid scintillation counting
What method is used to quantify gamma emitters like 125I?
Gamma counter
What is non-specific binding?
When a radioligand binds but not to the actual receptor and instead another part of the cell
How is a control sample prepared?
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
What can a saturation assay tell us about a tissue of interest?
Affinity (KD) and receptor density (Bmax)
What are two limitations of doing a saturation assay?
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
How can we compare the affinities of different drugs?
Use saturation assay to get KD value and compare affinity with another drug
KD and affinity are inversely proportional
How can we compare the selectivity of two drugs?
Use KD to compare the drugs affinities at two different receptors
What is the definition of an enzyme?
Biological catalysts that speed up the transformation of a substrate into a product
What are the two steps of the reaction between an enzyme and substrate?
Binding step that forms complex
Catalytic step that generates product
How is the Hill-Langmuir equation modified to fit enzyme activity
Michaelis Menton
Binding step of enzymes is equivalent to a drug and a receptor
