Receptor Theory Flashcards
What are the four classifications of receptors?
1) Ligand gated ion channel
2) G protein coupled receptors
3) Kinase linked receptors
4) Nuclear receptors
What model can be used to explain efficacy?
The ‘lock and key’ model - serveral keys may fit but not all of them will work and cause an effect
What is meant by affinity?
The strength of interaction - dependent on the forward rate of association as well as the backward rate of dissociation
High affinity means drugs associate strongly
How is occupancy calculated?
No of receptors occupied / total number of receptors
Can you rely on measuring response as a way of measuring occupancy?
No - although response may increase with occupancy the two are independent of each other.
Occupancy is related to affinity
Radio ligand binding assays can be used to measure what?
The affinity of a drug
Describe how to perform a radio ligand binding assay
1) Prepare cells eg use detergent and centrifugation
2) Aliquot membrane out onto filters
3) Add the radiolabel at different concentrations and equilibrate
4) When equilibrated remove the unbound drug by filtration - the bound drug remains attached to the the filter
5) Count the radioactivity of the filter
6) Use a radiowave counter and analyse data
How can non-specific binding be reduced?
Using anti-absorbants eg albumin/ collagen for peptides and o-catechol for catecholamines
How can you discriminate between specific and non-specific binding in a radio ligand binding assay?
In one set of tubes there will be the tissue and the radiolabelled ligand - this shows specific and non-specific binding
In the second set there will be the tissue with radiolabelled ligand and an excess of unlabelled radioligand
The radiolabelled ligand has no chance to compete with the unlabelled ligand for binding with the specific site so the radiolabelled ligand is completely displaced to non-specific binding sites
What properties must the radioligand have for the radio ligand binding assay?
1) it must be biologically active
2) it must be purified
3) Shouldn’t degrade
How can degradation of a radioligand be prevented?
1) By using free radical scavengers eg ethanol in the drug solution
2) Store at a low, but not freezing, temperature
3) Avoid light by storing in a dark bottle
4) Incorporation of antioxidant (ascorbic acid)
What are the advantages of 3H as a radioactive label?
The labelled product is indistinguishable from native compound
High specific activities can be obtained
Good stability when properly stored
Long half life -12.5 years
What are the disadvantages of 3H as a radioactive label?
Specialised labs are needed
Labelling is expensive and difficult
What are the advantages of 125I as a radioactive label?
If the compound has an aromatic hydroxyl group it can be incorporated at very high specific activities
Iodination is easy in most labs and is cheap
What are the disadvantages of 125I as a radioactive label?
More readily degraded
Biological activity of ligand can be reduced
Short half life - 67 days
How is the tissue with the bound ligand separated from the free ligand remaining in incubation media?
Usually by filtration and centrifugation
But in soluble binding other techniques are used; dialysis, column chromatography and precipitation/adsorption
The speed of separation between the tissue and the free ligand must be compatible with what?
The affinity of the ligand for the receptor ie a lower affinity requires faster and more efficient separation
Does Kd increase or decrease if affinity increases?
It decreases (Kd is the dissociation constant so if affinity is higher less drug will dissociate)
At what point will non-specific binding become an issue?
When the concentration of the ligand rises above 1000um
What is the scatchard equation?
B/F = (Bmax - B) / Kd B = specific bound ligand F = free radioligand (approx concentration added) Bmax = max number of binding sites Kd = Equilibrium binding constant/ affinity
What does the langmuir equation describe?
The relationship between receptor occupancy, affinity and the drug concentration
What is the Langmuir Equation?
Bound = Bmax Xa/ (Xa + Kd) = backward rate constant / forward rate constant
In a scatchard analysis the slope is inversely proportional to what?
The Kd/affinity
What is the Bmax (pmol/ mg protein)?
The maximum amount of drug which can specifically bind to the receptors in a membrane preparation. If ONE molecule binds to each receptor, then it also indicates the concentration of receptors in the tissue
Why do we need to measure receptors?
1) to define receptors eg different antagonist Kds = different receptors
2) To understand the rate of drug/ligand action
3) To find how many receptors are in a given tissue
4) To find out how receptor numbers change in disease
How can response to a drug be measured?
By bioassay
What is the EC50?
The effective concentration giving a 50% maximal response
What is meant by a receptor reserve?
Less than 100% of the receptors need to be occupied for there to be a maximal response
What are the steps involved in the contraction of guinea pig ilium in response to acetylcholine?
1) Ach binds to muscarinic receptor
2) Activation of G proteins
3) Stimulation of secondary messenger systems
4) Rise in intracellular machinery
5) Activation of contractile machinery
6) Contraction
What is the Hill equation?
Response = max.[Xa]^n/([Xa]^n+[EC50]^n max = maximum response eg amplitude of contraction Xa = concentration of agonist n = slope factor (often called the hill slope) EC50 = concentration of agonist evoking 50% of the response
n in the Hill equation is partially determined by what?
The number of molecules that must bind to the receptor for a response and is related to the steepness of the curve
What is meant by a more potent drug?
A drug which has a lower EC50
Why might pharmaceuticals want to develop partial agonists?
A partial agonist does not cause a full response but may be more potent than a full agonist.
They can help stop an overdose situation and prevent desensitisation of receptors
What is efficacy?
The measure of a single agonist-receptor complexes ability to generate a response
For a partial agonist what does Kd equal?
The EC50
What three properties determine the effect of a drug in a living system?
Specificity, Affinity and Efficacy
What is the efficacy of a full agonist?
1
What is the efficacy of an antagonist?
0
What is an inverse agonist and what is its efficacy?
An agonist which binds to receptors and stabilises them so they can’t consume an active configuration
Eg on GPCRs they bind to allosteric sites which alter the efficacy of another agonist
Their efficacy is below 0
What are the five classes of antagonists?
Chemical, Pharmacokinetic, Physical, Non-competitive and competitive
What is chemical antagonism?
Substances bind IN SOLUTION so the effects of the active drug is lost and the agonist can’t associate with the receptor
Eg Inactivation of heavy metals (mercury, lead and cadmium) where toxicity is reduced with the addition of a chelating agent (dimercaprol)
What is pharmacokinetic antagonism?
A reduction in the drug absorbed eg from the GI tract
For example drugs may inhibit gut motility which will reduce absorption via the oral route
Some drugs change the metabolism of other drugs eg antiobiotics can reduce the effect of warfarin
What is physiological antagonism?
Two drugs have opposing actions in the body eg noradrenaline raises arterial blood pressure whereas histamine lowers arterial blood pressure
What is non-competitive antagonism?
Where some steps in the process from receptor activation and response are blocked
eg it does not compete for the receptor active site
Example are L type channels found on smooth muscle- drugs used to lower blood pressure eg Nipedipine counteracts sympathetic activity
What is competitive antagonism?
Competes with the drug for the occupancy of the receptor
Most common type of antagonism
Binds reversibly and may be overcome by a big increase in agonist concentration
What is the dose ratio?
How many more times agonist is needed in the presence of an antagonist
Concentration of agonist in presence of antagonist/concentration of agonist in absence of antagonist
What does the schild analysis measure?
Antagonist affinity
How do you calculate dose ratio?
DR = ([Xb]/ Kd) +1 Xb = concentration of antagonist Kd = Antagonist affinity constant
What is irreversible competitive antagonism?
Antagonism which can not be reversed by washing the tissue
It is time dependent
Eg the effects of alkylating drug dibenamine on histamine responses in a guinea pig ileum
Why may the effect of a drug may decline overtime when given continously?
Receptors from the cell surface may be lost and endoctytosed
The receptor may change itself eg by phosphorylation
Exhaustation of mediators
Increased metabolic degredation or extrusion of the drug
Physiological adaptation
