L4 - Quantitative receptor pharmacology Pt1 Flashcards
define the key components of a competition binding experiment.
First, a saturation binding study measures the KA and RT of a nonselective, radiolabeled ligand (3H-QNB) to the receptors of interest
Following, a competition bending study uses a single concentration of the radioligand and varying concentrations of unlabelled receptor-subtype-specific ligands
These generate competition binding curves that determine the receptor subtype presence in the tissue.
Competition binding studies can be used to determine the ____ and ___ of receptors, but not ___
proportions (type) and densities (amount), function
Hypothetical competition binding experiment
A solution containing mAchRs from cells or tissue of interest is separated equally between tubes
equal amounts of non-selective radiolabelled ligand is added to each tube ([3H-QNB], in pM)
Increasing [Ligand A] is added to each tube, this is selective for a particular mAchR
Increasing [Ligand A] will reduce [3H-QNB] binding if that subtype is present (competition)
Solution is filtered which removes small, unbound drugs
Measure radioactivity in tube (cpm) (bound R + NSB) for each of the 12 different concentrations of cold, unbound ligand (e.g. Ligand A) used (Total binding).
demonstrate how competition binding experiments can be plotted and analysed
These experiments are analysed using a scatterplot graph where the radioactivity (cpm). on each tube is measured against the log of various [LA]
- at low [LA] there will be high cpm and as [LA] increases cpm will plateau to represent only NSB of radiolabeled ligand and this value can be subtracted from all previous values to give specific binding cpm (converted to %)
Demonstrate how specific binding values can be interpreted and analysed to characterize the receptor’s affinity and their subtype
the graph now shows the IC50 concentration (the [] required to inhibit 50% binding of a radioligand)
- this is not a measure of the affinity of the ligand for the receptor because it is proportional to the amount of radioligand added
The Cheng-Prusoff equation can be used now to determine affinity (Ki)
What is the Cheng-Prusoff equation?
An equation used to determine Ki (Affinity) where
Ki= IC50/1+(D/KA)
D = [ligand]
KA= Ka of radioligand (determined by Saturation Study)
- if low concentrations of radio ligand are used Ka becomes 0 and Ki = IC50
What is IC50
half maximal inhibitory concentration (IC)
How to use Ki values to identify receptors
the conversion of Ki to -log(Ki) gives the pKi. This value is an estimated value of affinity. This value is compared to pKi obtained from PURE populations of receptor subtypes when presented with Ligand A.
pKi values are compared to our estimate value to determine (by comparing affinity) the populations of receptor subtype inteh sample
Shapes of competition binding curves
Steep sigmoidal, Shallow sigmoidal and biphasic
- these reflect different patterns of
binding between the radioligand and the competing ligand
Steep sigmoidal curve
A curve is steep if 80% or more of specific binding occurs with one log unit of the logIC50
- find one log unit on either side of the logIC50 and subtract them
a STEEP curve indicates that you have either a pure population of
receptors OR a mixture of receptors for which it has equal affinity
Biphashic curve with a point of inflection
This indicates that more than one receptor is present for which the ligand A has markedly different affinities (-log Ki values differ by > 2 log units)
The point of inflection must be known to determine the -logKi of each receptor
- the POI represents the proportion of the receptor types present as a %
To determine the -logKi use the IC50 of each phase on either side of the POI
Receptor binding is represented as a percentage to each receptor e.g. 40% M1 & 60% to M3
SHALLOW curves
This curve reflects the binding of the ligand to more than one receptor for which it has slightly different affinities (-log Ki values differ by 0.5-2 log units)
the logIC50 value ± one log unit accounts for < 80% specific binding
Shallow sigmoidal curves cannot be interpreted reliably by eye–they need to be analysed more rigorously by fitting the data to computer-based models that describe binding to one or two, or more binding sites
