Molecular Pharmacology - Radioligand binding

Question 1

what is question 1 of the exam going to be on

Answer 1

quantitative analysis
it is compulsory

Question 2

what is the rationale behind resolving specific receptor binding

Answer 2

receptor binding is finite and saturable
non-specific binding is non-saturable

Question 3

how do you calculate specific binding

Answer 3

measure the non-specific non-saturable binding then measure the difference between that and the total binding
gives us the specific saturable binding

Question 4

what is the criterion for designing a radioligand binding assay

Answer 4

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

Question 5

what does a radioligand binding assay allow us to do

Answer 5

develop an estimate of ligand bound at different concentrations of ligand

Question 6

what is the criteria for identifying an appropriate ligand

Answer 6

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

Question 7

what isotopes do we usually use for radioligand binding assays

Answer 7

tritium
carbon-14
phosphorous-32
sulphur-35
iodine-131

Question 8

what is the becquerel (Bq)

Answer 8

Bq = number of disintegrations per second
usually per minute (d.p.m)

Question 9

how do we link the Bq back to a concentration of a ligand

Answer 9

we use the Curie constant

Question 10

what is the Curie (Ci) constant

Answer 10

2.22 x 10^23 d.p.m

Question 11

how do we link the Ci d.p.m to the concentration of ligand

Answer 11

we need to know its specific activity (SA)
SA = activity/mol
e.g. - 20Ci/mmol

Question 12

how do we measure radioactivity in solution

Answer 12

Scintillation counting - converts radioactivity into emitted light

Question 13

what are the different ways to separate bound from unbound ligand

Answer 13

filtration
sedimentation
centrifugation

Question 14

how is equilibrium dialysis used to

Answer 14

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

Question 15

how is competition binding used to distinguish specific vs non-specific binding

Answer 15

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

Question 16

what is IC50

Answer 16

the concentration of cold ligand that displaces 50% of the radioligand specific binding

Question 17

what information can we derive from a competition binding derived IC50 curve

Answer 17

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

Question 18

what is occupation theory and what assumption does this allow us to make

Answer 18

that the number of receptors occupied is proportional to the response
EC50 = Kd

Question 19

what is the problem with occupation theory

Answer 19

it is not true
does not need all the receptors to produce maximal response

Question 20

what are the disadvantages of radioligand binding

Answer 20

cannot distinguish between agonist/antagonist/partial agonists
non-physiological environment
long incubation times - may lead to receptor desensitisation

Question 21

what are the advantages of radioligand binding assays

Answer 21

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

Question 22

you need to redo lecture 3

Answer 22

how well do you understand it

Question 23

what are the advantages of a direct plot

Answer 23

does not require data transformation
requires no modelling
no distortion of data points

Question 24

what is 90%/95%/99% of the Bmax equal to in terms of Kd

Answer 24

90% of Bmax = 9 x Kd
95% 0f Bmax = 19 x Kd
99% of Bmax = 99 x Kd

Question 25

what does a curved scatchard plot indicate

Answer 25

that there is more than one binding site - site heterogeneity

Question 26

what will you need to be able to do for the exam

Answer 26

a scatchard plot
no hill plot - only tell what information it gives

Question 27

what is the IC50 dependent on

Answer 27

the concentration of radiolabelled ligand used and its Kd

Question 28

how do we convert the IC50 into an affinity constant

Answer 28

the Cheng-Prusoff equation corrects and calculates the equilibrium dissociation constant for the inhibitor