L6 - Receptor Theory II Flashcards
What is efficacy?
Full agonist will have an efficacy > 0
An antagonist will have an efficacy = 0
The maximum response achievable from an applied dose or agent.
What is occupancy?
The proportion of receptors occupied will vary with drug concentration
No drug present occupancy = 0
All receptors occupied occupancy = 1
In the case of agonist, the size of the response produced is proportional to?
Occupancy
This directly allows you to measure affinity
Radioligand binding assays of a ligand to a protein target are used to?
Measure affinity
The ligand could be
- Neurotransmitter, hormone, growth factor, cytokine, drugs, toxins
The protein target could be
- Receptors, ion channels, enzymes, carrier molecules
Radioligand binding assay steps
- Prepare protein sample
- Mix with drug on interest (drug needs to be made radioactive)
- Incubate to allow reaction to reach equilibrium
- Filter and rinse the drug away
- Count and analyse data
Non-specific binding
Most ligands bind non-specifically to tissue, filter paper, glass.
Measuring proportion of specific and non-specific binding is key
- Rinsing only removes unbound radioligand from incubation medium
Bound radioactivity = non specific + specific binding
Non-specific binding to filters & glass can be reduced by?
Anti-absorbents
- Albumin or collagen for peptides
- O-catechol for catecholamines
But this does not reduce non-specific binding to the tissue under study
Non-specific binding method
- Set up 2 rows of test tubes with increasing concentrations of the radioactive drug
- In one row add excess non-radioactive drug
- These will out compete the radioactive drug for the specific binding site (because they are in excess
The radioligand
The ligand must be biologically active since its binding to recognition site correlates with a response
The ligand must be extremely pure chemically
Labelling of the drug with radioactivity must achieve high specific activity to allow very low concentrations
Ways to solve degradation
Free-radical scavenger in drug solution
Store at low temperature
Avoiding light
Incorporation of antioxidant (e.g. ascorbic acid)
Advantages of 3H as a radio label
Labelled product indistinguishable from native compound
High specific activity
Good stability when properly stored
Long half life – 12 years
Disadvantages of 3H as a radio label
Specialised labs required
Labelling is expensive and difficult
Advantages of 125I as a radio label
If compound has an aromatic hydroxyl group can be incorporated at high specific activities
Iodination easy and cheap in most labs
Disadvantages of 125I as a radiolabel
More readily degraded
Biological activity of ligand can be reduced
Short half life – 67 days
Tissue and incubation conditions - tissue types
Isolated membranes Slices Synaptosomes Cultured cells Solubilized/purified receptors
Role of incubation
Must preserve integrity of both ligands and binding site
Additives used to protect tissue/ligand
- Protease inhibitors for peptides
- Antioxidants for ligand
Incubation conditions
Temperature usually around 0oC
Membrane protein concentration in range of 0.1-1 mg/ml with assay volumes 0.25-1 ml
Separating bound from free techniques
Filtration or centrifugation For soluble binding - Dialysis - Column chromatography - Precipitation/adsorption
Issues with separating bound from free
Major problem is rate of dissociation of ligand-receptor complex
Speed of separation must be compatible with affinity of ligand for receptor
Lower affinity (higher KD) requires faster and more efficient separation
Scatchard plot non-specific binding line
Linear- not on log
Non-linear when on log
Radioactive ligand and cold excess
Not saturable
Scatchard plot
Specific binding = total bound - nonspecific binding
Data is usually plotted on a semi-log scale
Radioligand binding is stereospecific
Scatchard plot total bound line
Increases rapidly then slows down
Radioactive ligand
Saturable because total number of receptors is limited
Scatchard plot specific binding line
Increases rapidly then plateaus (sigmoidal)
There will be a concentration of ligand where all the receptors will be labelled and the curve will plateau
The Scatchard equation
B/F = (Bmax- B)/KD
Langmuir equation
Describes the relationship between receptor occupancy, affinity and drug concentration
Langmuir equation/data is converted to a scatchard plot
Scatchard plot analysis - F
Free ligand = concentration of ligand added (mol/litre)
Scatchard plot analysis - B
Measured specific binding
Scatchard plot analysis - Bmax
(pmol/mg protein)
Maximum amount of drug which can bind specifically to the receptors
If one molecule binds to each receptor, then it indicates the concentration of receptors
Scatchard plot analysis - Kd
(mol/litre)
The dissociation constant for the radioligand determined by saturation analysis
The concentration of ligand which, at equilibrium, occupies 50% of the receptors
Low Kd
High affinity
High Kd
Low affinity
