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
what is non-specific binding in radioligand assays?
ligands may bind to not only the target receptors, but to other surfaces as well, such as other areas on the tissues, the filter, glass sides etc
binding to surfaces can be reduced by anti-absorbents such as collagen, albumin and o-catechol, but it does not reduce binding to other tissue areas
how can specific binding of the radioligand be calculated?
test tube A and B are identical in tissue (same amount of receptors) and have the same conc of radioactive drug:
test tube A: addition of huge excess of non-radioactive drug - non-radioactive drug out competes for radioactive drug for the specific binding
- radioactive drug is displaced from recognition sites by non-radioactive drug
test tube B: contains only radioactive drug
- radiolabelled ligands is used at low conc, but with high specific activity for detection
- radioactive drug binds non-specifically to target receptors
total bound (non-specific and specific binding from tube A) - non-specific binding tube B = specific binding
what governs an agonist inducing a response of a receptor?
efficacy:
- reversible reaction
- agonist stabilises receptor structure to favour signalling
- efficacy is measured using bioassay
what is the EC50?
the effective conc of an agonist giving 50% of maximal response in a specific tissue
- forms a sigmoidal curve due to concentration dependence, but binding and response curves may differ
what is the receptor reserve?
not 100% receptors need to be occupied to give a maximal 100% response
- due to amplification of signalling
- some tissues can get max response with <5% occupancy
what does the Hill equation measure?
the slope factor (Hill slope) is the measure of the no. molecules that need to bind to the receptor to activate it
any equation which produces a sigmoidal response
what does EC50 measure?
agonist potency:
- this will depend on affinity, efficacy and spare receptors
- Drug which has the highest potency is the drug which requires a lower conc to produce a 50% of max response
what are partial agonists?
- they are not able to produce the same maximum response as a full agonist
- in the presence of a full agonist, partial agonist acts like an antagonist and causes a right-shift in conc-response curve
how do partial agonists and full agonists differ?
- partial agonists have a lower efficacy than full agonists
- a partial agonist needs to achieve 100% occupancy to produce the maximum response that it is capable of
what is special about the KD and EC50 of partial agonists?
- the KD and EC50 of partial agonists are equal
what 3 properties determine the effect of a drug in a living system?
- specificity: interact with a structurally defined site/molecular structure
- affinity: agonists and antagonists’ ability to bind to receptor
- KD - efficacy: agonists’ ability to cause the response of a receptor
- full agonist has efficacy of 1
- antagonist has efficacy of 0
how can potency be measured?
- biological assays are designed to measure the relative potency of 2 preparations, usually a standard and an unknown
which ligand is majorly used for clinical drugs?
antagonists: prevent agonist resposne
what are the 5 classes of antagonism?
- chemical
- pharmacokinetic
- physiological
- non-competitive
- competitive
what is chemical antagonism?
- substances combine in solution so that the effects of the active drug is lost
- the antagonist chemically alters the agonist
e.g. chelating agent inactivates heavy metals such as mercury by reducing its toxicity
what is pharmacokinetic antagonism?
reduction in amount of drug absorbed, metabolised or excreted by another drug
how does a pharmacokinetic antagonist reduce the amount of an agonist being absorbed?
antagonist reduces the free concentration of drug at its target either by reducing drug absorption or by accelerating renal or hepatic elimination
e.g. decrease in agonist absorption at the GI tract:
- drugs that inhibit gut motility such as opiates will reduce absorption by the oral route of other drugs
how does a pharmacokinetic antagonist change the amount of an agonist being metabolised?
e.g. warfarin (anti-coagulant to thin blood and reduce heart attack)
- patients taking warfarin must be careful when taking antibiotics
- antibiotics can stimulate the metabolism of warfarin, causing the reduction of warfarin conc in the bloodstream
how does a pharmacokinetic antagonist change the amount of an agonist being excreted?
e.g. diuretics cause alteration of protein binding, filtration, urine flow and pH
- diuretics also inhibit tubular secretion
what is physiological antagonism?
the interaction of 2 drugs with opposing actions in the body:
- e.g. noradrenaline raises arterial blood pressure by acting on heart and blood vessels, while histamine lowers arterial pressure by causing vasodilation
- both NA and histamine act on the same receptor
what is non-competitive antagonism?
blocks some step in the process between agonist-receptor activation and response
- it does not compete with the agonist for the receptor
- it can block the agonist indirectly by inhibiting a signalling molecule, or by binding to another point on receptor to prevent function
give an example of a non-competitive antagonist:
Dihydropyridines: prevent opening of a voltage-gated Ca2+ channel to prevent contraction of muscle cells and reduce pressure to treat hypertension
what is competitive antagonism?
competes with agonist for occupancy of receptor:
- occupancy of agonist is reduced
- antagonist forms a complex with the receptor but does not stimulate any downstream signalling
what are the two types of competitive antagonism?
reversible
irreversible
only reversible antagonists can be overcome by increased agonist concentration
what dictates binding of a competitive antagonist?
by the equilibrium of the forward reaction and the reverse reaction
what is reversible competitive antagonism?
can be overcome by increasing the conc of the agonist
- e.g. effects of atropine on response to ACh in ileum
what change does a reversible competitive antagonist make to the conc-response curve?
- the curve causes a shift to the right
- the slope gradient and maximum however stays the same as the agonist can overcome it
- the higher the conc of the antagonist, the further the right shift
- EC50 increases with increase in competitive antagonist
what is the dose ratio?
how many more times the agonist is needed to overcome the presence of an antagonist:
DR = conc of agonist in presence of antagonist/conc of agonist in absence of antagonist
what is the Schild analysis?
quantifies the potency of a competitive antagonist:
- dose ratio is proportional to conc of added agonist / KD for that antagonist
- forms a linear straight line: 1/KD * x + 1
dose ratio of 2 means twice the amount of antagonist is needed to cause the same response
what does the Schild analysis allow us to determine?
the affinity constant of an antagonist by identifying the intersect of the x-axis
what is pA2?
intersect of x-axis * -1:
- pA2 is another way of expressing affintiy
- the bigger the pA2, the higher the affinity of the antagonist
how do partial agonists compare to competitive antagonists?
- they behave in the same way
- they both cause a right shift in the conc-response curve to a full agonist
however:
- partial agonist will have some agonist action, so the initial response increases, reaching a max equal to the max response of the partial agonist (in absence of full agonist)
- competitive antagonist cannot ever induce signalling
what are irreversible competitive antagonists?
antagonism that cannot be reversed by washing the tissue and increasing agonist conc:
- it is time-dependent
- if agonist conc is increased, the max response is decreased in presence of the irreversible antagonist (cannot be overcome)
- reaction does not reach equilibrium so we cannot measure KD using Schild analysis
