Lecture 2 - Drug-receptor interactions 1 (affinity) Flashcards
What is a receptors occupation governed by?
affinity
What is a receptors activation governed by?
Efficacy
What is affinity?
A way of describing the tightness of the fit between drug and receptor
What can you can a receptor when it has a ligand associated?
occupied
What determines the rate of reaction?
‘law of mass action’ - as well as the concentration of the drug (ligand) and receptor
Do the rules for affinity change depending on whether the drug is an agonist or an antagonist?
no
What is the efficacy of an antagonist?
0
What is a ligand?
A ligand is a molecule that binds receptor site on another molecule. This binding MAY or MAY NOT trigger a biological response
Affinity definition
The tendency for a drug to bind to a receptor is governed by its affinity.
What type of affinity do drugs of a high potency usually have?
A high affinity for their receptors
What is the definition of efficacy?
The tendency for a drug, once bound, to activate a receptor
What is KD?
a constant that defines the affinity of a drug for a receptor
What is KD expressed in?
Molarity
What is the forward rate of reaction?
K+1(A)*(R)
A = ligand
R = receptor
What is the reverse rate of reaction?
K-1(AR)
AR = ligand-receptor complexes
What is the equation at equilibrium?
K+1[A]*[R] = K-1[AR] = KD
What is occupancy?
proportion of receptors occupied will have vary with the drug concentration
What do you measure occupancy?
number of receptors occupied/total number of receptors (measured between 0 - 1)
Describe the relationship between occupancy and affinity
Direct relationship (inverse)
Why is a bioassay not a good way to determine affinity?
- Relationship between occupancy and response isn’t always linear
- Antagonist doesn’t generate resposne
What could be used to measure affinity?
Biochemical techniques
What are 4 experimental approaches used to measure drug affinity?
- Radioligand binding assay
- Fluorescence
polarisation assays - Surface Plasmon Resonance
- Isothermal Titration Calorimetry
- Computational modelling
What is a radioligand binding assay?
A radiolabeled drug is incubated with receptor-containing tissue or cells. The amount of radiolabel bound to the receptors is measured
What is an advantage of radiolabeled binding assays?
Sensitive - can be used with a wide range of receptors
What is a disadvantage of radiolabeled binding assays?
requires radioactive materials, may not be suitable for all receptors
What is fluorescence polarisation assays?
A fluorescently labeled drug is used. When it binds to a receptor, its rotational freedom decreases, leading to increased polarisation of emitted fluorescence
What is an advantage of fluorescence polarisation assays?
non-radioactive, high throughput, can be used for both small and large molecules
What is a disadvantage of fluorescence polarisation assays?
requires a fluorescently-labeled drug, may be less sensitive than radioligand binding assays
What is surface plasmon resonance?
Measures changes in the refractive index of a surface when molecules bind to it
What is an advantage of surface plasmon resonance?
real-time measurement, can be used for both small and large molecules
What is a disadvantage of surface plasmon resonance?
requires specialised equipment, may be less sensitive for low-affinity interactions
What is isothermal titration calorimetry?
Measures the heat released or absorbed during a binding interaction
What are the advantages of isothermal titration calorimetry?
provides thermodynamic parameters (enthalpy. entropy), can be used for both small and large molecules
What are the disadvantages of isothermal titration calorimetry?
requires specialised equipment, may be less sensitive for low-affinity interactions
What is computational modelling?
uses computational methods to PREDICT drug-receptor interactions based on molecular structures
What are advantages to computational modelling?
can provide insights into binding mechanisms, can be used to screen large libraries of compounds
What are disadvantages to computational modelling?
requires computational expertise, may not always accurately predict experimental results
What are examples of ligands used as radioligand binding assays?
- neurotransmitters
- hormones
- growth factors
-cytokines/chemokines - drugs
- toxins
What are examples of proteins used as radioligand binding assays?
- receptors
- ion channels
- enzymes
- carrier molecules
What does an increase in ligand lead to?
Increase in occupancy, but also increase in non-specific binding
Describe the process of a ligand-binding practical
- Tissue preparation + radioligand
- Mix and incubate
- Filter
- Rinse
- Count
- Analyse data
Describe the steps involved in source of receptors
Selected to contain the recognition sites (receptors of interest)
- can be isolated membranes, slices, synaptosomes, cultured cells or solubilized/purified receptors - e.g. from the total brain or specific brain region, or immortalized cell lines expressing the receptor.
Describe the steps involved in incubation
Must (try to) preserve integrity of ligands and receptors . “additives” used to protect tissue/ligand (protease inhibitors for peptides antioxidants if ligand is oxidizable.
Temperature: important parameter - usually low room to 0 degrees Celcius. Important as temperature affects enzyme activity.
Describe properties of the radioligand needed when measuring receptor affinity
Ligand must be biologically active, as it is suppose to correlate with a pharmacological action
PURITY - ligand must be extremely pure chemically
Degradation - major problem
Labelling - labelling of the drug with radioactivity must achieve very high specific activity to allow very low (“tracer”) concentrations
Describe ways to prevent degradation of radioligand
- Free-radical scavenger (e.g. ethanol) in drug solution
- Store at low (not freezing) temperature
- Avoiding light (dark bottles to store)
- Incorporation of antioxidants (ascorbic acid)
Describe the 2 choices of radio-labels
- Radioactive hydrogen (3H)
- Radioactive iodine (125I)
What are advantages of using radioactive hydrogen?
- labelled product indistinguishable from native compound
- high specific activities (>80 Ci/mmol) can be obtained
- good stability when properly stored
- Long half-life (12.5 years)
What are the disadvantages of using radioactive hydrogen (3H)?
- specialised labs required
- labelling is expensive& difficult
What are advantages of using radioactive iodine?
- if compound has an aromatic hydroxyl group (e.g. tyrosine residues in peptides) can be incorporated at very high specific activities (>2000 Ci/mmol)
- iodination easy in most labs and cheap
What disadvantages of using radioactive iodine (125I)?
- more readily degraded
- biological activity of ligand can be reduced (i.e. not functionally ‘invisible’)
- short half-life (67 days)
Describe the process of separating bound from free
Separation of bound and free ligand in incubation is done via filtration or centrifugation
- for soluble binding, other techniques are used - dialysis, column chromatography, precipitation/adsorption
- Major consideration 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/more efficient separation
Describe non-specific binding when measuring receptor affinity
- many ligands bind non-specifically to other proteins in the preparation, plastic, filter paper, glass
- non-specific binding to filters & glass can be reduced by ANTI-ABSORBANTS -e.g. albumin or collagen for peptides, o-catechol for catecholamines
- however, this doesn’t reduce non-specific binding to other proteins or lipids in the preparation.
- Measuring proportion of specific and non-specific binding is a KEY element to assay
- Rinsing only removed unbound - non-specific binding is determined by addition of EXCESS non-radioactive drug, as specific bound which is present will be displaced, however non-specific bound will remain
How do u find the specific radioligand binding curve?
take away the non-specific from the total:
Specific = Total bound - nonspecific binding
What measurement is concentration shown in?
nmol/L
What measurement is specificity bound shown in?
mol/mg
What type of scale is used to plot binding data?
semi-logarithmic scale
What is KD?
50% occupany
What is the equation for specific bound?
Specific bound =
amount of ligand specifically bound x total number of receptors //// concentration of ligand + KD (dissociation constant)
Describe why there is an inverse relationship between KD and affinity
Less drug required = better binding
Lower KD = higher affinity
What occurs in competition binding experiements?
Normal radioligand binding with a known drug.
In competition assays, increasing conc. of an unlabelled ligand is added together with a fixed concentration of a labelled ligand
e.g. Naloxone added to mu-opioid receptors bound with 3H-DAMGO.
increasing concentrations of Naloxone, the % of bound 3H-DAMGO decreases (displaced from receptor binding sites.
The concentration of Naloxone required to displace 50% bound 3H-DAMGO is the inhibition constant K, units Molar
New drug tested to see if it displaces the radioactive ligand.
Concentration of competitor to displace 50% of the known ligand with be the KD of the new drug.
