Molecular Pharmacology - Radioligand binding Flashcards
what is question 1 of the exam going to be on
quantitative analysis
it is compulsory
what is the rationale behind resolving specific receptor binding
receptor binding is finite and saturable
non-specific binding is non-saturable
how do you calculate specific binding
measure the non-specific non-saturable binding then measure the difference between that and the total binding
gives us the specific saturable binding
what is the criterion for designing a radioligand binding assay
1 - decide on the receptor source
2 - identify appropriate radioligands
3 - identify means to separate bound and unbound ligands
4 - identify means to distinguish specific and non-specific binding
what does a radioligand binding assay allow us to do
develop an estimate of ligand bound at different concentrations of ligand
what is the criteria for identifying an appropriate ligand
1 - high affinity to receptor
2 - slow dissociation kinetics
3 - prefer an antagonist over agonist - doesn’t cause changes in receptor structure
4 - high selectivity - specific
5 - compatible with labelling techniques
what isotopes do we usually use for radioligand binding assays
tritium
carbon-14
phosphorous-32
sulphur-35
iodine-131
what is the becquerel (Bq)
Bq = number of disintegrations per second
usually per minute (d.p.m)
how do we link the Bq back to a concentration of a ligand
we use the Curie constant
what is the Curie (Ci) constant
2.22 x 10^23 d.p.m
how do we link the Ci d.p.m to the concentration of ligand
we need to know its specific activity (SA)
SA = activity/mol
e.g. - 20Ci/mmol
how do we measure radioactivity in solution
Scintillation counting - converts radioactivity into emitted light
what are the different ways to separate bound from unbound ligand
filtration
sedimentation
centrifugation
how is equilibrium dialysis used to
if you are working with soluble receptors
you have 2 chambers separated by a semi-permeable membrane
allows free ligand to diffuse across leaving behind bound ligand and receptors
how is competition binding used to distinguish specific vs non-specific binding
we keep our radioligand of known affinity constant
increase concentration of unlabelled competitor (cold ligand/analogue)
to displace the non-specific binding of the radioligand
what is IC50
the concentration of cold ligand that displaces 50% of the radioligand specific binding
what information can we derive from a competition binding derived IC50 curve
affinity of a specific receptor to ligand (Kd)
pharmacological profile of the receptor
number of receptor sites in a tissue (Bmax)
characteristics of the drug-receptor interaction
what is occupation theory and what assumption does this allow us to make
that the number of receptors occupied is proportional to the response
EC50 = Kd
what is the problem with occupation theory
it is not true
does not need all the receptors to produce maximal response
what are the disadvantages of radioligand binding
cannot distinguish between agonist/antagonist/partial agonists
non-physiological environment
long incubation times - may lead to receptor desensitisation
what are the advantages of radioligand binding assays
no pharmacokinetic confounds
direct measure of Kd
direct measure of Bmax
defines molecular characteristics of drug-receptor interaction
suitable for high throughput screening
defines pharmacological profile of receptor
you need to redo lecture 3
how well do you understand it
what are the advantages of a direct plot
does not require data transformation
requires no modelling
no distortion of data points
what is 90%/95%/99% of the Bmax equal to in terms of Kd
90% of Bmax = 9 x Kd
95% 0f Bmax = 19 x Kd
99% of Bmax = 99 x Kd
