Receptors (L5-8) Flashcards
What do most drugs target?
Proteins like receptors, enzymes and transporters like carriers or channels
What is the difference between a receptor and drug target?
Receptors are proteins who’s function is to recognise and respond to endogenous chemical signals. Drug targets are other macromolecules which drugs interact with to produce the effect.
What are the different types of receptor families?
Type 1 - ligand gated ion channels (ionotropic receptors)- they have 4/5 transmembrane spanning domains. They’re activated by ligands
Type 2 - G protein coupled receptors (metabotropic because they use energy) Have multiple binding domains with a G-protein coupled domain.
Type 3 - Kinase-linked receptors. Have a binding domain on each terminal. Binding of an extracelllar ligand causes enzymatic activity on the IC side (catalytic domain)
Type 4 - Nuclear receptors - found in the cytosol until a ligand binds then they move into the nucleus - ussually have DNA binding domains (zinc fingers)
What does the activation of a receptor depend on?
The activation of a receptor by the agonist depends on the efficacy of the drug. A higher efficacy elicits a larger response.
What what is meant by the occupancy of a receptor and what does it depend on?
The proportion of receptors occupied which varies with concentration. (the number of receptors occupied / total number of receptors) The affinity of the drug to the receptor - the higher the affinity, the higher the occupancy will be. Its a ratio so varies between 0 (none occupied) and 1 (all occupied)
How can you measure the occupancy?
Using a tagged agonist - one with GFP on it which fluoresces when its bound to a receptor and then you can measure the amount of florescence. A higher result = a higher occupancy.
Explain non-specific binding of ligands
Most ligands bind non-specifically to other things like filter paper, and glass. his can be reduced using anti-absorbants e.g. albumin or collagen for peptides or o-catechol for catecholamines. But this does not reduce non-specific binding to the tissue under study. Measuring proportions of specific and non-specific binding is a key element to an assay. Rinsing only removes unbound radio ligand (the marker) from the incubation medium.
Explain the properties a radio ligand must have to make it useful for experiments
Must be biologically active because its binding is suppossed to correlate with a pharmacological action. The ligand must be extrememly pure chemically and not degrade - you can get around this by using a free radical scavenger e.g. ethanol in you drug solution. Stroing it at low temps and out of direct sunlight, and incorporating an antioxidant e.g. ascorbic acid aslo helps with this.
Labelling of the drug with readioactivity must achieve a very high specific activity ( activity per unit mas) to all very low tracer concentrations.
What are the different radio labels that could be used to study a drug?
3H - Labelled product is indistinguishable from the native compund, highe specific activity can be obtained. It has a good stability when stored properly and has a long half life (12.5 yrs). However, it is expensive and difficult to use.
125I - Used if the compound has an aromatic hydroxyl group (e.g. tyrosines of peptides). It can be incoprporated at very high specific activities but the biological activity of the ligand may be reduced and it have a musch shorter half life (67 days)
How can you separate the bound ligand from the free ligand in the incubation media?
by centrifugation then filtration is the ligand is insoluble. For soluble ligands (and solubilised receptors) you have to use other techniques like dialysis, column chromatography and precipitation/adsorption
The main problem is the rate of dissociation of the ligand-receptor complexes must be compatible with the affinity of a ligand for a receptor. A lower affinity (Higher KD) requires a faster, more efficient separation.
What is a scatchard plot?
The plot of the ratio of the concentrations of a bound ligand to an unbound ligand. It is a method for analysing data freely for reversible ligand/receptor binding interactions where you can’t use a tagged ligand because it doesn’t stay put - help you work out the affinity constant
Specific binding =total bound - non-specific binding. It shows a plateau for specific binding because the receptors become saturated, but not for non-specific because the receptor can continue to bind to other things like the glass and tissue.
Data is plotted on a semi log scale ( The conc of the ligand is on a log scale) as this converst the retangular hyperbola into a sigmoid curve
What is the scatchard equation?
B/F (specifically bound/free ligand) = Bmax (max no. of binding sites)- B / Kd (eq.binding constant) - so basically ratio of no. of receptors occupied to ligand added = total no. of receptors - specifially bound divided by dissociation constant - give you a measure of affinity
How do you use the scatchard plot to work out the affinity constant?
B/F is what is plotted on the Y axis (bound radio ligand/free radioligand(approx the conc of the free ligand added in mol/litre bc the amount that ia actually bound and not free is negligible) Bmax is the max amount of drug which can bind specifically to the receptors in the preparation (also indicates the conc of receptors in the tissue) Bmax is the X intercept bc B/F = 0 when all the receptor is bound. Therefore, the slope is the inverse of - Kd (Kd is found using saturation analysis, it is the conc of the ligand which at equilibrium occupies 50% of the receptor
How does Kd shift on an conc Vs occupancy graph?
A lower Kd is more the the left because the drug has a higher affinity and therefore needs less conc for a high occupancy. A high Kd means a lower affinity because a high conc is needed for the same occupancy.
How can you use binding data to learn about the selectivity of different ligands?
If you try the same drug on 2 different tissues, you will see a different occupancy for each. (One will show a lower affinity). Whereas a non-selective ligand will have the same occupancy in both tissues.
