Receptor Theory Flashcards
how does binding and activation differ between agonists and antagonists?
- both can bind to a receptor
- only AGONISTS can activate receptors to induce signalling and a response
what feature of a drug governs its occupation of a receptor?
AFFINITY:
- drug and receptor associate in forward reaction
- drug and receptor disassociate in reverse reaction
rate of forward reaction = k+1
rate of reverse reaction = k-1
what feature of a drug governs its activation and response of a receptor?
EFFICACY:
- antagonists have an efficacy of 0
- full agonists have an efficacy of 1
- antagonists can prevent the action of agonists
what are partial agonists?
- agonists which cannot produce a full response from a receptor even when occupying it
- they have an intermediate efficacy between 0 and 1
what is KD?
dissociation constant that defines the affinity of a drug to a receptor
- drug-receptor interactions are reversible and involve interaction of 2 molecules
what is the forward rate of reaction equation?
k+1 = A * R
A = conc of ligand, R = conc of receptors
- second-order reaction as it depends on conc of 2 molecules
- as conc of either increases, the reaction is pushed forward so more product is made
what is the reverse rate of reaction equation?
k-1 = AR
AR = conc of ligand-receptor complexes formed
- first-order reaction as it only depends on conc of AR
what is the dissociation constant (KD) equation?
KD = AR / A*R
KD in molarity
direct measure of affinity
if high affinity, KD is small.
if low affinity, KD is high.
what is occupancy?
the proportion of receptors occupied vary with drug concentration
occupancy is governed by affinity
occupancy varies between 0 (no drug present) and 1 (all receptors occupied)
what is the equation for occupancy?
occupancy = no. receptors occupied / total no. receptors
if a high affinity drug is applied to a cell, how will this affect the KD and occupancy?
KD will be small
occupancy will be high
what are radioligand binding assays?
measurement of the binding of a ligand to a protein target to find its affinity
- drug is labelled with radioactive isotope, and radioactivity is measured at the tissue
what is the process of radioligand binding?
- prepare cells/membranes via detergent treatment and centrifugation
- divide out membranes onto filters
- add radiolabel at different concs and equilibrate
- when equilibrated, remove unbound drug by filtration
- bound drug will remain attached to filter - count radioactivity of filter
how are tissues selected in radioligand binding assays?
- selected to obtain the recognition receptor sites of interest
- can be isolated membranes, slices, synaptosomes, cultured cells or solubilised/purified receptors
e.g. from total brain or specific brain region, or immortalised cell lines expressing the receptor of interest
how are tissues incubated in radioligand binding assays?
the integrity of both ligands and binding sites must be preserved to ensure protein targets aren’t denatured:
- additives are used to protect the tissue/ligand e.g. protease inhibitors for peptides, antioxidants if ligand is oxidisable e.g. catecholamines
- temperature: usually between 0C and room temp
what is the criteria used for the radioligand in the assay?
- it must be biologically active as its binding to a receptor must correlate with pharmaceutical action
- radioactivity must not change molecular structure of drug
- ligand must be chemically pure
- avoid degradation of ligand using free-radical scavenger in drug solution (ethanol), store at low temp + avoid light, use antioxidant (ascorbic acid)
- labelling must achieve high specific activity to allow low tracer concs
what are the two possible choices of radiolabels?
Hydrogen isotope 3H
iodine isotope 125I
what are the ads/disads of the hydrogen isotope in radiolabelling?
ads:
- Labelled product indistinguishable from native compound
- High specific activities can be obtained
- Good stability when stored properly
- Long half-life (12.5 years)
disads:
- Specialised labs required
- Labelling is expensive and difficult
what are the ads/disads of the iodine isotope in radiolabelling?
ads:
- If compound has aromatic hydroxyl group (e.g. tyrosine residues in peptides) it can be incorporated at very high specific activities
- Iodination easy in most labs and cheap
disads:
- More easily degenerated
- Biological activity of ligand can be reduced
- Short half-life (67 days)
how are tissue-bound ligands separated from the free-ligands in radioligand binding assays?
filtration or centrifugation
soluble binding needs other techniques such as dialysis, column chromatography, precipitation
what is the problem with separation of bound and free ligands in radioligand assays
rate of dissociation of ligand-receptor complexes:
- speed of separation must be compatible with affinity of ligand for the receptor
- lower affinity (higher KD) requires more efficient separation