pharmacology - measuring drug action at receptors

features of what the following reaction represents D + R ←> DR (dose response)

• goverened by binding affinity

• quantified in terms of KD, the concentration of drug required to occupt 50% of receptors at equillibrium

features of what the following reaction represents DR ←> DR* (dose response)

• goverened by efficacy, the ability of drugs to activiate the receptor once bound

• difficult to quantify

• drug action described in terms of high (full agonist), low (partial agonist) or zero (competitive agonist) efficacy

how is affinity measured under radioligand binding assays

1. label the drug with a radioactive isotope e.g. 3H or 32P)

2. this allows us to detect and measure the amount of drug bound in tissue samples by detecting amount of radioactivity present in the tissue and the affinity of the drug for sites

3. homogenise a brain and divide equally amongst two sets of test tubes

4. incubate the first set of samples with varying concentrations of drug order from lowest concentration with lowest binding to highest

5. filter and wash samples to leave behind only radiolabelled drug bound brain tissue

6. measure the amount of radioactivity (bound drug) per sample

7. the second set of test tubes is used to determine max binding sites by separating specific and non specific binding

8. incubate second test tube set with the same concentration of radiolabelled drug plus a large excess of non radioactive ‘cold’ drug

9. filter and wash samples to leave behind only radiolabelled drug bound to brain tissue

10. measure amount of radioactivity (bound drug) per sample

explain the graph of a radioligand binding assay would look

given the finite number of receptors, the amount of binding should plateau once all receptors have a drug molecule bound i.e. saturation occurs

• if no plateu occurs, there is off target binding and drug begins to bind to another receptor (likely proteins)

what graph does the proportion of binding of drug to receptor produce

rectangular hyperbola that reaches a maximum as all the available recptors are occupied

D + R ←> DR

what is total binding

occurs in radioligand binding assays where no plateau is reached in binding (no saturation of receptors) this is because specific and non specific binding occurs

in radioligand binding assay experiments, what graph would the second set of test tubes produce and explain it

this represents only non specific binding since all the receptor sites are occupied by the ‘cold’ drug so are not free to bind to the radioactive drug

when conducting a radioligand binding assay, how to we measure of specific binding

total binding - non specific binding

→ this forms a saturation plot as the specific binding saturated as the receptor sites become full

→ from this information, we can calculate Bmax (maximum number of receptors per tissue) and KD (affinity of the radiolabelled drug)

outline what a reversible competitive antagonist is

drugs competing for the same receptor as an agonist

e.g. pancuronium, cetirizine, propranolol

inhibits the effects of a neurotransmittr or hormone

their effects are overcome by increasing the concentration of the agonist i.e. the blockade is surmountable

explain what a reversible competitive antagonist affinity from concentration-response curves would look like and graph it

reversible competitive antagonists produce a parallel shift to the right of the agonist log concentration vs response curve

the graph with the biggest shift to the right is the strongest antagonist meaning more agonist must be added to surmount it

outline what an irreversible competitive antagonist action would look like on a graph

they dont produce a parallel shift

however they still produce a shift in the agonist log concentration-response curve, it is not parallel

i.e. the inhibition they produce cannot be overcome by increasing the agonist concentration it is not surmountable

explain how antagonist affinity can be measured

• the extent to which the agonist curve is shifted by the antagonist (i.e. the amount of agonist you must add to overcome the blocking action of the antagonist) is related to the affinity of the antagonist for the receptor

• the shift in the curves can be measured using the dose-ratio i.e. the ratio of the concentration of agonists producing the same sized response in the presence and absense of the antagonist

• for a reversible competitive antagonist, the dose ratio should increase linearly with the concentration of the antagonist (i.e. a parallel shift will form)

define pA2

the negative logarithm of the polar concentration of antagonist that produces a dose ratio 2

• this is how the affinity of a reversible competitive antagonist is quantified

how is pA2 quantified

1. construct a log concentration response curve for the agonist in the presence of several concentrations of antagonist

2. thus a schild analysis can also tell us whether the antagonist is reversible and competitive

3. if you dont see a parallel shift you cant continue

what is the schild equation and how is it used

log (dose ratio -1) = log (antagonist concentration) - log KB

KB is the equilibrium dissociation consant (equivalent to KD) for an antagonist measured using this technique

for a reversible competitive antagonist a plot of log (dose ratio -1) versus log antagonist concentration (a Schild plot) should have a gradient of 1 and the x-intercept at (logKB)

for a reversible competitive antagonist

pA2 = pKB = -logKB

outline the steps in producing a schild plot

1. construct (log) ceoncentration-response curves to the antagonist in the presence of several concentrations of antagonists

2. from the curves read the logEC50 values for the agonist in the absense and presence of the different concentrations of antagonists

3. calculate the EC50 values for the agonists in the absense and presence of the different concenctrations of the antagonists

when using the schild equation, if the slop does not equal 1, what does this tell us about the nature of the antagonist

it is not a reversible competitive antagonist