agonists and functional analysis

Question 1

what is the main from of antagonism and what two other types of antagonism

Answer 1

▪ Receptor antagonism is the most common form (by far) But you may come across other types e.g.

• Physiological antagonists referring to things that produce opposing actions by a separate molecular mechanism E.g. noradrenaline and acetylcholine on heart rate
• Antagonism of the stimulating messenger · E.g. antibody therapies that inactivate protein growth factors

Question 2

what are the four different flavours of receptor antagonists and what do they mean

Answer 2

1. Competitive vs Non-competitive
2. Reversible vs irreversible

• Competitive antagonists are just antagonists that bind at the same site as the agonist
• Non-competitive antagonists bind at a different site from the agonist
• Reversible antagonists bind non-covalently to our receptor, so they can both Associate and dissociate from the receptor
• irreversible antagonists which covalently bind to the receptor and can’t dissociate

Question 3

what type of antagonism can be surmounted with higher concentrations of agonist

Answer 3

reversible antagonism

Question 4

what type of antagonist is causing this rightward shift (decrease) in agonist potency i.e. EC50

Answer 4

this is caused by a reversible competitive antagonist

Question 5

what are the two possible explanations for the antagonist causing this concentration-response curve

Answer 5

1. Non-surmountable antagonism

• The antagonist reduces the maximal agonist response
• This can occur when the agonist is irreversible
• Irreversible agonists form covalent bonds with the receptor
• E.g. phenoxybenzamine at a-adrenoreceptors

2. Non competitive antagonist

• Non-competitive antagonists bind an allosteric site, separate from the agonist orthosteric site

Question 6

explain why non-competitive antagonists are advantageous therapeutically?

Answer 6

non-competitive antagonists are advantageous therapeutically

because regardless of how much agonist you have it can still bring maximum response down, and this is because it binds to a different site so it doesn’t have to compete with the agonist

Question 7

what is the concetration ratio?

referring to comparing antagonist vs control CRCs (concentration response curves)

state the equation to find it

Answer 7

• the size of the shift between the control and the presence of the antagonist

Question 8

Once we know there Concentration ratio (CR) we can find out the affinity of the antagonist (remember: NOT the agonist) using there gaddum equation

state the gaddum equation

Answer 8

Question 9

is ipratropium a surmountable antagonist in this example? - explain why

could we therefore use the gaddum equation with this information?

Answer 9

Yes (to both)

the absence and presence of ipratropium have the same maximal response therefore we have everything we need to use the gaddum equation

Question 10

what analysis techinque can we use if we have CR values for several antagonist concentrations and we want to interept this

hint: it is a rearrangement of the gaddum equation

Answer 10

schild analysis

Question 11

what are the steps to do a schild analysis to estimate the antagonist affinity

hint: the picture gives a clue for the first step

Answer 11

1. calculate the CR betwen each of the curves
2. use the values for log [antagonist] and plot a graph for log [CR-1] (y axis) against log[antagonist] (x axis)
3. the intercept of the line should be an estimate for the antagonist affinity

Question 12

the Schild plot should be a straight line with a gradient of 1, if it isn’t then this means there could be a problem

give two examples for what could be wrong

Answer 12

1. the antagonist is not competitive
2. the assay was not in equilibrium

Question 13

The Schild plot analysis often uses a different nomeculature

what is the log[Kd] (antagonist affinity) referred to as

Answer 13

pA₂ = log[Kd]